Compounds of the inventions of guanylyl cyclase C

ABSTRACT

Guanylin cyclase C compound of the inventions are disclosed. Conjugated compounds comprising guanylin cyclase C compound of the inventions conjugated to detectable or therapeutic moieties are disclosed. Methods of detecting, imaging and treating cancer and treating diarrhea are disclosed.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority to U.S. Provisional Application No.60/583,039 filed Jun. 25, 2004, which is herein incorporated byreference in its entirety.

FIELD OF THE INVENTION

The present invention relates to peptides and peptide analogues bind tothe cellular receptor protein guanylyl cyclase C (GCC). In someembodiments, the peptides and peptide analogues are agonists andactivate the signaling pathway that is activated by the binding of thenatural GCC ligands to GCC. In some embodiments, the peptides andpeptide analogues block binding of natural ligands of GCC but do notactivate the signaling pathway activated by the binding of the naturalGCC ligands to GCC.

BACKGROUND OF THE INVENTION

Gastrointestinal malignancies, including adenocarcinoma of theesophagus, stomach, colon, and rectum, are a leading cause of cancer andcancer-related mortality worldwide. GC-C is a transmembrane receptorexpressed only on the apical membranes of intestinal enterocytes innormal, healthy adults and by primary and metastatic tumor cells ofgastric, esophageal and colorectal adenocarcinoma. Conzelmann, M., etal., Cytokeratin 20 and guanylyl cyclase C mRNA is largely present inlymph node and liver specimens of colorectal cancer patients. Int JCancer, 2003. 107(4): p. 617-28; Cagir, B., et al., Guanylyl cyclase Cmessenger RNA is a biomarker for recurrent stage II colorectal cancer.Ann Intern Med, 1999. 131(11): p. 805-12; Bustin, S. A., et al.,Quantification of cytokeratin 20, carcinoembryonic antigen and guanylylcyclase C mRNA levels in lymph nodes may not predict treatment failurein colorectal cancer patients. Int J Cancer, 2004. 108(3): p. 412-7;Pearlman, J. M., et al., A splice variant of the transcript for guanylylcyclase C is expressed in human colon and colorectal cancer cells. DigDis Sci, 2000. 45(2): p. 298-305; Waldman, S. A., et al., Use ofguanylyl cyclase C for detecting micrometastases in lymph nodes ofpatients with colon cancer. Dis Colon Rectum, 1998. 41(3): p. 310-5;Salto-Tellez, M., et al., Intrinsic variability in the detection ofmicrometastases in lymph nodes for re-staging of colorectal cancer.effect of individual markers and tissue samples. Eur J Cancer, 2003.39(9): p. 1234-41; Chen, G., et al., Detection of occult metastasis inlymph nodes from colorectal cancer patients: a multiple-marker reversetranscriptase-polymerase chain reaction study. Dis Colon Rectum, 2004.47(5): p. 679-86; Notarnicola, M., et al., K-ras and p53 mutations inDNA extracted from colonic epithelial cells exfoliated in faeces ofpatients with colorectal cancer. Dig Liver Dis, 2000. 32(2): p. 131-6;Hugues, M., et al., Identification and characterization of a new familyof high-affinity receptors for Escherichia coli heat-stable enterotoxinin rat intestinal membranes. Biochemistry, 1991. 30(44): p. 10738-45;Carrithers, S. L., et al., Escherichia coli heat-stable enterotoxinreceptors. A novel marker for colorectal tumors. Dis Colon Rectum, 1996.39(2): p. 171-81; Carrithers, S. L., et al., Guanylyl cyclase C is aselective marker for metastatic colorectal tumors in humanextraintestinal tissues. Proc Natl Acad Sci USA, 1996. 93(25): p.14827-32; Carrithers, S. L., et al., Escherichia coli heat-stable toxinreceptors in human colonic tumors. Gastroenterology, 1994. 107(6): p.1653-61; Bustin, S. A., et al., Detection of cytokeratins 19/20 andguanylyl cyclase C in peripheral blood of colorectal cancer patients. BrJ Cancer, 1999. 79(11-12): p. 1813-20; Pitari, G. M., et al., Guanylylcyclase C agonists regulate progression through the cell cycle of humancolon carcinoma cells. Proc Natl Acad Sci USA, 2001. 98(14): p. 7846-51;Tien, Y. W., et al., Simultaneous detection of colonic epithelial cellsin portal venous and peripheral blood during colorectal cancer surgery.Dis Colon Rectum, 2002. 45(1): p. 23-9; Park, J., et al., Ectopicexpression of guanylyl cyclase C in adenocarcinomas of the esophagus andstomach. Cancer Epidemiol Biomarkers Prev, 2002. 11(8): p. 739-44;Pitari, G. M., et al., Bacterial enterotoxins are associated withresistance to colon cancer. Proc Natl Acad Sci USA, 2003. 100(5): p.2695-9; Vlems, F. A., et al., Investigations for a multi-marker RT-PCRto improve sensitivity of disseminated tumor cell detection. AnticancerRes, 2003. 23(11A): p. 179-86; Tien, Y. W., et al., The role ofgelatinase in hepatic metastasis of colorectal cancer. Clin Cancer Res,2003. 9(13): p. 4891-6; Chen, W. S., et al., Impact of Circulating FreeTumor Cells in the Peripheral Blood of Colorectal Cancer Patients duringLaparoscopic Surgery. World J Surg, 2004; and Tien, Y. W., et al.,Intravasation-Related Metastatic Factors in Colorectal Cancer. TumourBiol, 2004. 25(1-2): p. 48-55, which are each incorporated herein byreference.

GCC has been identified previously as a specific marker and target forGI malignancies. Identification of the presence of GCC in ectopic sites,for example lymph nodes or blood, can be used as a marker to identifythe presence of occult micrometastases of esophageal, gastric, colonic,or rectal cancers. One method to detect the presence of GCC is toquantify the amount of binding of a natural ligand of GCC, such as ST,in tissues. Also, GCC appears to be a highly-specific target to whichcan be directed novel imaging and therapeutic agents to treat metastaticesophageal, gastric, colonic, and rectal cancers. Indeed, diagnostic ortherapeutic agents can be targeted to GCC-expressing tumors by theirconjugation to GCC ligands such as ST.

U.S. Pat. No. 5,518,888 issued May 21, 1996 to Waldman, PCT applicationPCT/US94/12232 filed Oct. 26, 1994, U.S. application Ser. No. 08/467,920filed Jun. 6, 1995, and U.S. application Ser. No. 08/583,447 filed Jan.5, 1996, which are each incorporated herein by reference, disclose thatmetastasized colorectal tumors can be targeted for delivery of activecompounds by targeting ST receptors (also referred to as guanylincyclase C or GCC). The presence of ST receptors on cells outside of theintestinal tract as a marker for colorectal cancer allows for thescreening, identification and treatment of individuals with metastasizedcolorectal tumors. ST receptors may also be used to target delivery ofgene therapeutics and antisense compounds to colorectal cells.

U.S. Pat. No. 5,601,990 issued Feb. 11, 1997 to Waldman, PCT applicationPCT/US94/12232 filed Oct. 26, 1994, and PCT application PCT/US97/07467filed May 2, 1997, which are each incorporated herein by reference,disclose that detection of evidence of expression of ST receptors insamples of tissue and body fluid from outside the intestinal trackindicate metastasized colorectal cancer.

U.S. Patent Application Publication No. 20010029019 published Oct. 11,2001, which is each incorporated herein by reference, discloses thatprimary and metastasized stomach and esophageal cancer tumors can betargeted for delivery of active compounds by targeting GCC. GCC servesas a marker for primary and metastasized stomach and esophageal cancerand allows for the screening, identification and treatment ofindividuals with primary and metastasized stomach and esophageal cancer.GCC is also be used to target delivery of gene therapeutics andantisense compounds to primary and metastasized stomach and esophagealcancer.

GCC regulates the balance of proliferation and differentiation of theepithelium in intestine. The intestinal epithelium is dynamic, with awell-defined vertical axis extending from the crypt depths, in the wallof the intestine, to the tips of villi which project out into the lumenof the intestine. Epithelial cells are “born” at the bottom of crypts asdaughter cells produced by intestinal stem cells. These daughter cellscontinue to divide (proliferate) and their progeny migrate up the wallof the crypt toward the tip of the villus. Along this migration, thecells shift from proliferation to differentiation to becomefully-functional mature enterocytes with the capacity to perform thenormal functions of the gut including digestion, absorption andsecretion. Once at the tip, these cells slough off into the lumen of theintestine and die. Thus, the intestinal epithelium turns over ˜everythree days. GCC and its endogenous ligands appear to be one of thefactors that shifts epithelial cells from proliferation todifferentiation along the crypt-villus axis. Indeed, GCC ligands inhibitthe proliferation of these cells and change their gene expressionpattern to a more terminally-differentiated state.

The GC-C protein has not been detected in any non-cancerous tissueoutside of the intestine and is only found on tumors of gastrointestinalorigin. Of particular importance to this proposal, the detection of GC-CmRNA has not been reported in any colorectal cancer metastatic tissues,i.e. non-colorectal tissue where metastatic colorectal cancer is oftendetected. For normal individuals, the presence of GC-C mRNA has not beendescribed in the published literature for the metastatic sites ofcolorectal, gastric or esophageal cancer (i.e., liver, lung, bone,brain), but it has been described in the proximal tubule cells of thekidney (Sindice, A., et al., Guanylin, uroguanylin, and heat-stableenterotoxin activate guanylate cyclase C and/or a pertussistoxin-sensitive G protein in human proximal tubule cells. J Biol Chem,2002. 277(20): p. 17758-64, which is incorporated herein by reference),exocrine duct cells of the pancreas (Kulaksiz, H., et al., Guanylin inthe human pancreas' a novel luminocrine regulatory pathway ofelectrolyte secretion via cGMP and CFTR in the ductal system. HistochemCell Biol, 2001. 115(2): p. 131-45, which is incorporated herein byreference), submandibular glands (Kulaksiz, H., et al., Guanylin andfunctional coupling proteins in the human salivary glands and glandtumors: expression, cellular localization, and target membrane domains.Am J Pathol, 2002. 161(2): p. 655-64, which is incorporated herein byreference), the bile duct (Kulaksiz, H., et al., Guanylin regulateschloride secretion in the human gallbladder via the bile fluid.Gastroenterology, 2004. 126(3): p. 732-40. which is incorporated hereinby reference) and at trace levels in CD34+ stem cells (Fava, T. A., etal., Ectopic expression of guanylyl cyclase C in CD34+ progenitor cellsin peripheral blood. J Clin Oncol, 2001. 19(19): p. 3951-9). A commontheme for each of these anatomical locations, except CD34+ cells, isthat GC-C is separated from the blood and lymphatic systems by tightjunctions analogous to that found in the intestine.

Diarrheal diseases are the fourth leading cause of mortality worldwide,responsible for about 20 million deaths each year. Such diseases are theleading cause of pediatric mortality worldwide, particularly affectingchildren under 5 years of age. Further, diarrheal diseases areresponsible for ˜25% of the growth retardation observed in childrenraised in under-developed, compared to developed, nations. One majorcause of diarrheal disease are organisms producing heat-stableenterotoxins (STs), a family of structurally-related peptides producedby different organisms including, but not limited to, E. coli, Yersinia,Enterobacter, and Vibrio. This family of structurally-related STpeptides is homologous to the endogenous peptides guanylin anduroguanylin produced in mammalian intestine. ST-producing organisms area major cause of endemic diarrhea in under-developed countries, theleading cause of travelers' diarrhea, and the leading cause of diarrhealdisease in agriculturally-important animal populations (scours) indeveloped and under-developed countries. It is estimated that the annualincidence of ST-induced diarrheal disease numbers in the billions inanimals and humans. ST induces diarrhea by binding to GCC, which isselectively expressed in brush border membranes of intestinal epithelialcells and the presumed receptor for the endogenous ligands guanylin anduroguanylin. Interaction of ST, or the endogenous ligands guanylin anduroguanylin, with GCC activates that receptor resulting in theproduction of intracellular cyclic GMP. Cyclic GMP, through a signalingcascade induces the secretion of salt and water into the lumen of theintestine, resulting in diarrhea. It has been suggested that onefunction for the endogenous ligands guanylin and uroguanylin in normalphysiology is the regulation of fluid and electrolyte homeostasis inintestine, and the hydration of intestinal contents (e.g. stool).

Over the past 20 to 30 years, attempts have been made to design ligandsthat antagonize GCC. Such a compound of the invention would be useful inthe detection of GCC on cells, as a targeted diagnostic and therapeuticagent in cases of GI malignancies, and in the treatment of diarrhealdiseases of animals and humans. Moreover, such a compound of theinvention might have application in cases requiring intestinaladaptation, wherein the epithelium requires rapid regeneration followingan insult, for example chemical or ischemic damage. Previousstructure-function studies of GCC ligands were unrevealing with respectto the discriminating the structural determinants required for receptorbinding from those required for agonist activation (e.g. cyclic GMPproduction).

There is a need for compounds that bind to GCC and activate the GCCsignal pathway and there is a need for compounds which bind to GCC butdo not activate the GCC signal pathway.

SUMMARY OF THE INVENTION

The present invention provides compounds that bind to GCC. Endogenousligands of GCC and enterotoxins known to bind to GCC are characterizedas having two disulphide bonds (forming Loop B and Loop C) or threedisulphide bonds (forming Loop A, Loop B and Loop C). The compounds ofthe invention have either Loop A or Loop B but not both. The compoundsof the invention can be conjugated to active agents such as detectablelabels, imaging agents or therapeutic compounds and used to bind to GCCwhich is a cancer marker for metastasized colorectal cancer and forprimary and metastasized esophageal and stomach cancer.

Some compounds of the invention that have Loop A are GCC compound of theinventions. They bind to GCC but do not activate the GCC pathway. Thoseare useful to treat undesirable activation of the GCC pathway such asthat associated with diarrhea such as travelers diarrhea brought on byexposure to bacterial enterotoxin. Some aspects of the present inventionrelate to these compounds and to methods of using them.

Some compounds of the invention that have Loop B are GCC compound of theinventions, some are GCC agonists and some are GCC prodrugs which areGCC compound of the inventions but when processed in vivo are convertedto agonists. Some compounds that have Loop B are GCC compound of theinventions; they bind to GCC but do not activate the GCC pathway. Theyare useful to treat undesirable activation of the GCC pathway such asthat associated with diarrhea such as travelers diarrhea brought on byexposure to bacterial enterotoxin. Some aspects of the present inventionrelate to these compounds and to methods of using them.

Some compounds that have Loop B are GCC agonists; they bind to GCC andactivate the GCC pathway. They are useful to treat cancer, particularlymetastasized colorectal cancer and primary and metastasized esophagealand stomach cancer, as well as prevent metastasis and activate the GCCpathway to induce defecation such when an individual is constipated orimpacted. Some aspects of the present invention relate to thesecompounds and to methods of using them.

Some compounds that have Loop B are GCC prodrugs. These compounds bindto GCC but do not activate the GCC pathway when intact. When thesecompounds are processed, such as when processed in vivo by endogenous oradministered enzymes that degrade its C terminal residues, they becomeGCC agonists. These compounds are useful to treat cancer, particularlymetastasized colorectal cancer and primary and metastasized esophagealand stomach cancer, as well as prevent metastasis and activate the GCCpathway to induce defecation such when an individual is constipated orimpacted. They have the advantage of having delayed activity andcontrollable conversion by using enzyme inhibitors to prevent thedegradation. When used in conjunction with enzyme inhibitors thatprevent degradation, their conversion can be controlled or preventedwhereby they act as compound of the inventions which make them useful toinhibit GCC activation

The present invention relates to compounds having a structure accordingto formula (I)R1-R2-R3-R4-R5-R6-R7  (I)wherein:

-   -   R1 is combination of 1-50 natural amino acids, 1-50 blocked        amino acids, 1-50 synthetic amino acids, 1-50 derivatized amino        acids; 1-50 amino acid mimetics, a 1-50 alkyls, one or more 1-50        substituted alkyls, one or more 1-50 aryls, one or more 1-50        substituted aryls, one or more 1-50 alkylaryls, one or more 1-50        substituted alkylaryls or combinations thereof;    -   R2 is a natural amino acid, blocked amino acid, synthetic amino        acid, derivatized amino acid; an amino acid mimetic;    -   R3 is a natural amino acid, blocked amino acid, synthetic amino        acid, derivatized amino acid or amino acid mimetic that        crosslinks with R8;    -   R4 is one or two natural amino acids, one or two blocked amino        acids, one or two synthetic amino acids, one or two derivatized        amino acids, one or two amino acid mimetics, or a dipeptide        mimetic or combinations thereof, wherein R4 forms a 3₁₀-helix        turn group linking R3 to R5;    -   R5 is a natural amino acid, blocked amino acid, synthetic amino        acid, derivatized amino acid or amino acid mimetic that does not        crosslink with R9    -   R6 is a natural amino acid, blocked amino acid, synthetic amino        acid, derivatized amino acid, or amino acid mimetic;    -   R7 is one, two or three natural amino acids, one, two or three        blocked amino acids, one, two or three amino acid mimetics, or a        tripeptide mimetic or combinations thereof, wherein R7 forms a        beta turn group linking R6 to R8    -   R8 is a natural amino acid, blocked amino acid, synthetic amino        acid, derivatized amino acid or amino acid mimetic that        crosslinks with R3;    -   R9 is a combination of 1-50 natural amino acids, 1-50 blocked        amino acids, 1-50 synthetic amino acids, 1-50 derivatized amino        acids; 1-50 amino acid mimetics, a 1-50 alkyls, one or more 1-50        substituted alkyls, one or more 1-50 aryls, one or more 1-50        substituted aryls, one or more 1-50 alkylaryls, one or more 1-50        substituted alkylaryls or combinations thereof that does not        crosslink to R5; provided the structure is not:        C-hC-E-L-A-C-N-P-A-X; hC;        C-E-L-A-X-N-P-A-C;        C-C-E-L-A-C-N-P-A-C;        C-C-E-L-A-C-N-P-A-C-T-G-A;        Y-Ca-C-E-L-F-Ca-N-P-A-C;        Y-C-C-E-L-mA-C-N-P-A-C;        Y-C-C-E-mL-A-C-N-P-A-C;        C-C-E-L-Cm-C-N-P-A-C-A-G-Cm;        C-C-E-L-Ca-C-N-P-A-C-A-G-Ca; or        X-Ca-E-L-A-hC-N-P-A-Ca;        wherein

-   hC is Homocysteine;

-   X is Alanine linked to hC via a covalent CH2-S bond between an Ala    side chain and an hC side chain;

-   Cm is Cysteine(S-methoxybenzyl); and

-   Ca is Cysteine(S-acetamidomethyl).

The present invention also relates to compounds that are Loop A compoundof the inventions. The present invention relates to compounds havingFormula (II):R201-R202-R203-R204-R205-R206-R207 wherein:  (II)

-   -   R201 is absent or 1-50 natural amino acids, 1-50 blocked amino        acids, 1-50 synthetic amino acids, 1-50 derivatized amino acids;        1-50 amino acid mimetics, a 1-50 alkyls, one or more 1-50        substituted alkyls, one or more 1-50 aryls, one or more 1-50        substituted aryls, one or more 1-50 alkylaryls, one or more 1-50        substituted alkylaryls or combinations thereof;    -   R202 is absent, or is a natural amino acid, blocked amino acid,        synthetic amino acid, derivatized amino acid or an amino acid        mimetic which does not crosslink to any other amino acid, or is        a natural amino acid, blocked amino acid, synthetic amino acid,        derivatized amino acid or amino acid mimetic that crosslinks        with R205 or R206, wherein if R202 is absent or a natural amino        acid, blocked amino acid, synthetic amino acid, derivatized        amino acid or an amino acid mimetic which does not crosslink to        any other amino acid then R203 is a natural amino acid, blocked        amino acid, synthetic amino acid, derivatized amino acid or        amino acid mimetic that crosslinks with R205 or R206;    -   R203 is absent, or is a natural amino acid, blocked amino acid,        synthetic amino acid, derivatized amino acid or an amino acid        mimetic which does not crosslink to any other amino acid, or if        R202 is absent or is a natural amino acid, blocked amino acid,        synthetic amino acid, derivatized amino acid or an amino acid        mimetic which does not crosslink to any other amino acid then        R203 is a natural amino acid, blocked amino acid, synthetic        amino acid, derivatized amino acid or amino acid mimetic that        crosslinks with R205 or R206;    -   R204 is one or two natural amino acids, one or two blocked amino        acids, one or two synthetic amino acids, one or two derivatized        amino acids, one or two amino acid mimetics, or a dipeptide        mimetic or combinations thereof, wherein R204 forms a 3₁₀-helix        turn group linking R203 to R205;    -   R205 is absent, or is a natural amino acid, blocked amino acid,        synthetic amino acid, derivatized amino acid or an amino acid        mimetic which does not crosslink to any other amino acid, or if        R206 is absent or is a natural amino acid, blocked amino acid,        synthetic amino acid, derivatized amino acid or an amino acid        mimetic which does not crosslink to any other amino acid then        R205 is a natural amino acid, blocked amino acid, synthetic        amino acid, derivatized amino acid or amino acid mimetic that        crosslinks with R202 or R203;    -   R206 is absent, or is a natural amino acid, blocked amino acid,        synthetic amino acid, derivatized amino acid or an amino acid        mimetic which does not crosslink to any other amino acid, or is        a natural amino acid, blocked amino acid, synthetic amino acid,        derivatized amino acid or amino acid mimetic that crosslinks        with R202 or R203, wherein if R206 is absent or a natural amino        acid, blocked amino acid, synthetic amino acid, derivatized        amino acid or an amino acid mimetic which does not crosslink to        any other amino acid then R205 is a natural amino acid, blocked        amino acid, synthetic amino acid, derivatized amino acid or        amino acid mimetic that crosslinks with R202 or R203; and    -   R207 is absent or 1-50 natural amino acids, 1-50 blocked amino        acids, 1-50 synthetic amino acids, 1-50 derivatized amino acids;        1-50 amino acid mimetics, a 1-50 alkyls, one or more 1-50        substituted alkyls, one or more 1-50 aryls, one or more 1-50        substituted aryls, one or more 1-50 alkylaryls, one or more 1-50        substituted alkylaryls or combinations thereof.

The present invention also relates to compounds that are Loop Bcompounds. The present invention relates to compounds having Formula(III):R301-R302-R303-R304-R305-R306-R307-R308-R309  (III)wherein:

-   -   R301 is combination of 1-50 natural amino acids, 1-50 blocked        amino acids, 1-50 synthetic amino acids, 1-50 derivatized amino        acids; 1-50 amino acid mimetics, a 1-50 alkyls, one or more 1-50        substituted alkyls, one or more 1-50 aryls, one or more 1-50        substituted aryls, one or more 1-50 alkylaryls, one or more 1-50        substituted alkylaryls or combinations thereof;    -   R302 is a natural amino acid, blocked amino acid, synthetic        amino acid, derivatized amino acid, or an amino acid mimetic        wherein R302 may crosslink with R306;    -   R303 is a natural amino acid, blocked amino acid, synthetic        amino acid, derivatized amino acid or amino acid mimetic that        crosslinks with R308;    -   R304 is one or two natural amino acids, one or two blocked amino        acids, one or two synthetic amino acids, one or two derivatized        amino acids, one or two amino acid mimetics, or a dipeptide        mimetic or combinations thereof, wherein R304 forms a 3₁₀-helix        turn group linking R303 to R305;    -   R305 is a natural amino acid, blocked amino acid, synthetic        amino acid, derivatized amino acid or amino acid mimetic that        does not crosslink with R309    -   R306 is a natural amino acid, blocked amino acid, synthetic        amino acid, derivatized amino acid, or amino acid mimetic        wherein R306 may crosslink with R302;    -   R307 is one, two or three natural amino acids, one, two or three        blocked amino acids, one, two or three amino acid mimetics, or a        tripeptide mimetic or combinations thereof, wherein R307 forms a        beta turn group linking R306 to R308    -   R308 is a natural amino acid, blocked amino acid, synthetic        amino acid, derivatized amino acid or amino acid mimetic that        crosslinks with R303;    -   R309 is absent or 1-50 natural amino acids, 1-50 blocked amino        acids, 1-50 synthetic amino acids, 1-50 derivatized amino acids;        1-50 amino acid mimetics, a 1-50 alkyls, one or more 1-50        substituted alkyls, one or more 1-50 aryls, one or more 1-50        substituted aryls, one or more 1-50 alkylaryls, one or more 1-50        substituted alkylaryls or combinations thereof that does not        crosslink to R305;    -   wherein the structure of the compounds is not:        C-hC-E-L-A-C-N-P-A-X; hC        C-E-L-A-X-N-P-A-C;        C-C-E-L-A-C-N-P-A-C;        C-C-E-L-A-C-N-P-A-C-T-G-A;        Y-Ca-C-E-L-F-Ca-N-P-A-C;        Y-C-C-E-L-mA-C-N-P-A-C;        Y-C-C-E-mL-A-C-N-P-A-C;        C-C-E-L-Cm-C-N-P-A-C-A-G-Cm;        C-C-E-L-Ca-C-N-P-A-C-A-G-Ca; or        X-Ca-E-L-A-hC-N-P-A-Ca;        wherein

-   hC is Homocysteine;

-   X is Alanine linked to hC via a covalent CH2-S bond between an Ala    side chain and an hC side chain;

-   Cm is Cysteine(S-methoxybenzyl); and

-   Ca is Cysteine(S-acetamidomethyl).

The present invention further relates to compounds having Formula (IV):R401-R402-R403-R404-R405-R406-R407-R408-R409  (IV)wherein:

-   -   R401 is combination of 1-50 natural amino acids, 1-50 blocked        amino acids, 1-50 synthetic amino acids, 1-50 derivatized amino        acids; 1-50 amino acid mimetics, a 1-50 alkyls, one or more 1-50        substituted alkyls, one or more 1-50 aryls, one or more 1-50        substituted aryls, one or more 1-50 alkylaryls, one or more 1-50        substituted alkylaryls or combinations thereof;    -   R402 is a natural amino acid, blocked amino acid, synthetic        amino acid, derivatized amino acid, or an amino acid mimetic        wherein R402 may crosslink with R406;    -   R403 is a natural amino acid, blocked amino acid, synthetic        amino acid, derivatized amino acid or amino acid mimetic that        crosslinks with R408;    -   R404 is one or two natural amino acids, one or two blocked amino        acids, one or two synthetic amino acids, one or two derivatized        amino acids, one or two amino acid mimetics, or a dipeptide        mimetic or combinations thereof, wherein R404 forms a 3₁₀-helix        turn group linking R403 to R405;    -   R405 is a natural amino acid, blocked amino acid, synthetic        amino acid, derivatized amino acid or amino acid mimetic that        does not crosslink with R409    -   R406 is a natural amino acid, blocked amino acid, synthetic        amino acid, derivatized amino acid, or amino acid mimetic        wherein R406 may crosslink with R402;    -   R407 is one, two or three natural amino acids, one, two or three        blocked amino acids, one, two or three amino acid mimetics, or a        tripeptide mimetic or combinations thereof, wherein R407 forms a        beta turn group linking R406 to R408    -   R408 is a natural amino acid, blocked amino acid, synthetic        amino acid, derivatized amino acid or amino acid mimetic that        crosslinks with R403;    -   R409 is absent;    -   wherein the structure of the compounds is not:        C-hC-E-L-A-C-N-P-A-X-hC        C-E-L-A-X-N-P-A-C;        C-C-E-L-A-C-N-P-A-C;        Y-Ca-C-E-L-F-Ca-N-P-A-C;        Y-C-C-E-L-mA-C-N-P-A-C;        Y-C-C-E-mL-A-C-N-P-A-C;        C-C-E-L-Cm-C-N-P-A-C-A-G-Cm;        C-C-E-L-Ca-C-N-P-A-C-A-G-Ca; or        X-Ca-E-L-A-hC-N-P-A-Ca;        wherein

-   hC is Homocysteine;

-   X is Alanine linked to hC via a covalent CH2-S bond between an Ala    side chain and an hC side chain;

-   Cm is Cysteine(S-methoxybenzyl); and

-   Ca is Cysteine(S-acetamidomethyl).

The present invention also relates to compounds that are Loop Bprodrugs. The present invention relates to compounds having Formula (V):

Formula (V):R501-R502-R503-R504-R505-R506-R507-R508-R509  (V)wherein:

-   -   R501 is combination of 1-50 natural amino acids, 1-50 blocked        amino acids, 1-50 synthetic amino acids, 1-50 derivatized amino        acids; 1-50 amino acid mimetics, a 1-50 alkyls, one or more 1-50        substituted alkyls, one or more 1-50 aryls, one or more 1-50        substituted aryls, one or more 1-50 alkylaryls, one or more 1-50        substituted alkylaryls or combinations thereof;    -   R502 is a natural amino acid, blocked amino acid, synthetic        amino acid, derivatized amino acid, or an amino acid mimetic        wherein R502 may crosslink with R506;    -   R503 is a natural amino acid, blocked amino acid, synthetic        amino acid, derivatized amino acid or amino acid mimetic that        crosslinks with R508;    -   R504 is one or two natural amino acids, one or two blocked amino        acids, one or two synthetic amino acids, one or two derivatized        amino acids, one or two amino acid mimetics, or a dipeptide        mimetic or combinations thereof, wherein R504 forms a 3₁₀-helix        turn group linking R503 to R505;    -   R505 is a natural amino acid, blocked amino acid, synthetic        amino acid, derivatized amino acid or amino acid mimetic that        does not crosslink with R509    -   R506 is a natural amino acid, blocked amino acid, synthetic        amino acid, derivatized amino acid, or amino acid mimetic        wherein R506 may crosslink with R502;    -   R507 is one, two or three natural amino acids, one, two or three        blocked amino acids, one, two or three amino acid mimetics, or a        tripeptide mimetic or combinations thereof, wherein R507 forms a        beta turn group linking R506 to R508    -   R508 is a natural amino acid, blocked amino acid, synthetic        amino acid, derivatized amino acid or amino acid mimetic that        crosslinks with R503;    -   R509 is 1-50 natural amino acids, 1-50 blocked amino acids, 1-50        synthetic amino acids, 1-50 derivatized amino acids; 1-50 amino        acid mimetics, a 1-50 alkyls, one or more 1-50 substituted        alkyls, one or more 1-50 aryls, one or more 1-50 substituted        aryls, one or more 1-50 alkylaryls, one or more 1-50 substituted        alkylaryls or combinations thereof that does not crosslink to        R505;    -   wherein the compound is degradable in vivo and the structure of        the compounds is not C-C-E-L-A-C-N-P-A-C-T-G-A.

The present invention also relates to compounds that are Loop B compoundof the inventions. The present invention relates to compounds havingFormula (VI):R601-R602-R603-R604-R605-R606-R607-R608-R609  (VI)wherein:

-   -   R601 is combination of 1-50 natural amino acids, 1-50 blocked        amino acids, 1-50 synthetic amino acids, 1-50 derivatized amino        acids; 1-50 amino acid mimetics, a 1-50 alkyls, one or more 1-50        substituted alkyls, one or more 1-50 aryls, one or more 1-50        substituted aryls, one or more 1-50 alkylaryls, one or more 1-50        substituted alkylaryls or combinations thereof;    -   R602 is a natural amino acid, blocked amino acid, synthetic        amino acid, derivatized amino acid, or an amino acid mimetic        wherein R602 may crosslink with R606;    -   R603 is a natural amino acid, blocked amino acid, synthetic        amino acid, derivatized amino acid or amino acid mimetic that        crosslinks with R608;    -   R604 is one or two natural amino acids, one or two blocked amino        acids, one or two synthetic amino acids, one or two derivatized        amino acids, one or two amino acid mimetics, or a dipeptide        mimetic or combinations thereof, wherein R604 forms a 3₁₀-helix        turn group linking R603 to R605;    -   R605 is a natural amino acid, blocked amino acid, synthetic        amino acid, derivatized amino acid or amino acid mimetic that        does not crosslink with R609    -   R606 is a natural amino acid, blocked amino acid, synthetic        amino acid, derivatized amino acid, or amino acid mimetic        wherein R606 may crosslink with R602;    -   R607 is one, two or three natural amino acids, one, two or three        blocked amino acids, one, two or three amino acid mimetics, or a        tripeptide mimetic or combinations thereof, wherein R607 forms a        beta turn group linking R606 to R608    -   R608 is a natural amino acid, blocked amino acid, synthetic        amino acid, derivatized amino acid or amino acid mimetic that        crosslinks with R603;    -   R609 is 1-50 natural amino acids, 1-50 blocked amino acids, 1-50        synthetic amino acids, 1-50 derivatized amino acids; 1-50 amino        acid mimetics, a 1-50 alkyls, one or more 1-50 substituted        alkyls, one or more 1-50 aryls, one or more 1-50 substituted        aryls, one or more 1-50 alkylaryls, one or more 1-50 substituted        alkylaryls or combinations thereof that does not crosslink to        R605 and is non-degradable in vivo.

The present invention further relates to conjugated compounds thatcomprise a compound of a formula selected from the group consisting of:Formula (I), Formula (II), Formula (III), Formula (IV), Formula (V), andFormula (VI), that is conjugated to an active moiety which may be aradionuclide, an enzyme, a fluorescent label, a metal chelating group, achemiluminescent label, a bioluminescent label, a chemotherapeutic, atoxin, an inactive prodrug, a radiosensitizing agent, a photodynamicagent, a nucleic acid molecule or combinations thereof.

The present invention further relates to compositions comprisingliposomes that comprise compounds of the invention, conjugated orunconjugated, in combination with an active agent selected from thegroup consisting of: a radionuclide, an enzyme, a fluorescent label, ametal chelating group, a chemiluminescent label, a bioluminescent label,a chemotherapeutic, a toxin, an inactive prodrug, a radiosensitizingagent, a photodynamic agent, a nucleic acid molecule or combinationsthereof.

The present invention further relates to compositions that comprisecompounds of the invention in combination with a chemotherapeutic, atoxin, or combinations thereof.

The present invention relates to methods of diagnosing cancercharacterized by expression of GCC in an individual. The methodscomprising the step of detecting GCC in an extraintestinal sample froman individual by contacting the sample or portions thereof with suchcompound and detecting the presence of the compound bound to the sample.

The present invention relates to methods of imaging cancer characterizedby expression of GCC in an individual. The methods comprise the step ofadministering to the individual such a conjugated compound in whichactive moiety of the conjugated compound is a detectable label. Theaccumulation of the conjugated compound at a site on the individual'sbody is detected.

The present invention further relates to methods of treating cancercharacterized by expression of GCC in an individual. The methodscomprise the step of administering to the individual such a conjugatedcompound in which the active moiety kills or inhibits replication ofcells to which it binds.

The present invention further relates to method of treating anindividual who has enterotoxin mediated diarrhea or who is at risk ofcontracting enterotoxin mediated diarrhea. The methods comprise the stepof administering to said individual a compound with a structureaccording to Formula (II) or Formula (VI).

The present invention further relates to method of treating anindividual who has enterotoxin mediated diarrhea or who is at risk ofcontracting enterotoxin mediated diarrhea. The methods comprise the stepof administering to said individual a compound with a structureaccording to Formula (I), Formula (III), Formula (IV) or Formula (V)under conditions such that the C terminal is not degradable such thatthe compound is converted to an agonist.

The present invention further relates to method of treating anindividual who has cancer or who is at risk of developing cancer. Themethods comprise the step of administering to said individual a compoundwith a structure according to Formula (I) Formula (III), Formula (IV) orFormula (V) under conditions such that the C terminal is degradable suchthat the compound is converted to an agonist. In some embodiments, thecompounds are administered in combination with a chemotherapeutic agentor a toxin.

The present invention further relates to method of treating anindividual who has cancer or who is at risk of developing cancer. Themethods comprise the step of administering to said individual a compoundwith a structure according to Formula (II). In some embodiments, thecompounds are administered in combination with a chemotherapeutic agentor a toxin.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 show data: Effect of Hydrophobicity at Residue 9;

FIG. 2: Lead Compound of the inventions (comparing ST(4-14)Phe9,Cys(Mob)5,10Mono, ST(4-14)NMeLeu8 and ST(4-14)NMeAla9 at 1 and 25 microMwith and without ST)

FIG. 3: Lead GC-C Compound of the inventions (comparing ST(4-14)Phe9,Cys(Mob)5, 10Mono, ST(4-14)NMeLeu8, ST(4-14)NMeAla9 and ST(4-14) Ala9,17at 1 and 25 microM)

FIG. 4: Screen for GC-C Compound of the inventions (comparing 16different compound of the inventions at 1 microM)

FIG. 5: A table containing raw data of the 16 different compound of theinventions at 1 microM and 25 microM

FIG. 6: Effect of residue 9 on ST antagonist efficacy.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS OF THE INVENTION

Definitions:

As used herein, the following terms shall have the following meanings:

As used herein, the terms “antagonist,” “antagonist compounds,”“antagonists of the invention” is meant to refer to compounds which bindto GCC and block GCC binding to natural ligands but do not activate theGCC pathway.

As used herein, the terms “agonist,” “agonist compounds,” “agonists ofthe invention” is meant to refer to compounds which bind to GCC andblock GCC binding to natural ligands and activate the GCC pathway.

As used herein, the term “natural ligands” is meant to refer to heatstabile enterotoxins as well as endogenously produced GCC ligandsguanylin and uroguanylin.

As used herein, the term “active agent” is meant to refer to compoundsthat are therapeutic agents or imaging agents.

As used herein, the term “therapeutic agent” is meant to refer tochemotherapeutics, toxins, radiotherapeutics, targeting agents orradiosensitizing agents.

As used herein, the term “chemotherapeutic” is meant to refer tocompounds that, when contacted with and/or incorporated into a cell,produce an effect on the cell including causing the death of the cell,inhibiting cell division or inducing differentiation.

As used herein, the term “toxin” is meant to refer to compounds that,when contacted with and/or incorporated into a cell, produce the deathof the cell.

As used herein, the term “radiotherapeutic” is meant to refer toradionuclides which when contacted with and/or incorporated into a cell,produce the death of the cell.

As used herein, the term “targeting agent” is meant to refer compoundswhich can be bound by and or react with other compounds. Targetingagents may be used to deliver chemotherapeutics, toxins, enzymes,radiotherapeutics, antibodies or imaging agents to cells that havetargeting agents associated with them and/or to convert or otherwisetransform or enhance co-administered active agents. A targeting agentmay include a moiety that constitutes a first agent that is localized tothe cell which when contacted with a second agent either is converted toa third agent which has a desired activity or causes the conversion ofthe second agent into an agent with a desired activity. The result isthe localized agent facilitates exposure of an agent with a desiredactivity to the cancer cell.

As used herein, the term “radiosensitizing agent” is meant to refer toagents which increase the susceptibility of cells to the damagingeffects of ionizing radiation. A radiosensitizing agent permits lowerdoses of radiation to be administered and still provide atherapeutically effective dose.

As used herein, the term “imaging agent” is meant to refer to compoundswhich can be detected.

As used herein, the term “active moiety” is meant to refer to theportion of a conjugated compound that constitutes an active agent.

As used herein, the terms “conjugated compound” and “conjugatedcomposition” are used interchangeably and meant to refer to a compoundwhich comprises a compound of the invention as a GCC binding moiety andan active moiety. The conjugated compound is which is capable of bindingto GCC. Conjugated compounds according to the present invention comprisea portion which constitutes compound of the invention and a portionwhich constitutes an active agent. Thus, conjugated compounds accordingto the present invention are capable of specifically binding to GCC andinclude a portion which is a therapeutic agent or imaging agent.Conjugated compositions may comprise linkers and/or molecules that serveas spacers between the moieties.

As used herein, the terms “linker”, “linking agent”, “conjugatingagent”, “coupling agent”, “condensation reagent” and “bifunctionallinker” are used interchangeably and are meant to refer to moleculargroups which are used to attach the compound of the invention and theactive agent to thus form the conjugated compound.

As used herein, the term “colorectal cancer” is meant to include thewell-accepted medical definition that defines colorectal cancer as amedical condition characterized by cancer of cells of the intestinaltract below the small intestine (i.e. the large intestine (colon),including the cecum, ascending colon, transverse colon, descendingcolon, and sigmoid colon, and rectum). Additionally, as used herein, theterm “colorectal cancer” is meant to further include medical conditionswhich are characterized by cancer of cells of the duodenum and smallintestine (jejunum and ileum). The definition of colorectal cancer usedherein is more expansive than the common medical definition but isprovided as such since the cells of the duodenum and small intestinealso contain GCC.

As used herein, the term “stomach cancer” is meant to include thewell-accepted medical definition that defines stomach cancer as amedical condition characterized by cancer of cells of the stomach.

As used herein, the term “esophageal cancer” is meant to include thewell-accepted medical definition that defines esophageal cancer as amedical condition characterized by cancer of cells of the esophagus.

As used herein, the term “metastasis” is meant to refer to the processin which cancer cells originating in one organ or part of the bodyrelocate to another part of the body and continue to replicate.Metastasized cells subsequently form tumors which may furthermetastasize. Metastasis thus refers to the spread of cancer from thepart of the body where it originally occurs to other parts of the body.

As used herein, the term “metastasized colorectal cancer cells” is meantto refer to colorectal cancer cells which have metastasized.Metastasized colorectal cancer cells localized in a part of the bodyother than the duodenum, small intestine (jejunum and ileum), largeintestine (colon), including the cecum, ascending colon, transversecolon, descending colon, and siginoid colon, and rectum.

As used herein, the term “metastasized stomach cancer cells” is meant torefer to stomach cancer cells which have metastasized. Metastasizedstomach cancer cells localized in a part of the body other than thestomach.

As used herein, the term “metastasized esophageal cancer cells” is meantto refer to colorectal cancer cells which have metastasized.Metastasized esophageal cancer cells localized in a part of the bodyother than the esophagus.

As used herein, the term “non-colorectal sample” and “extra-intestinalsample” are used interchangeably and meant to refer to a sample oftissue or body fluid from a source other than colorectal tissue. In somepreferred embodiments, the non-colorectal sample is a sample of tissuesuch as lymph nodes. In some preferred embodiments, the non-colorectalsample is a sample of extra-intestinal tissue which is an adenocarcinomaof unconfirmed origin. In some preferred embodiments, the non-colorectalsample is a blood sample.

As used herein, “an individual suffering from an adenocarcinoma ofunconfirmed origin” is meant to refer to an individual who has a tumorin which the origin has not been definitively identified.

As used herein, “an individual is suspected of being susceptible tocolorectal, stomach or esophageal cancer” is meant to refer to anindividual who is at a particular risk of developing colorectal, stomachor esophageal cancer. Examples of individuals at a particular risk ofdeveloping colorectal, stomach or esophageal cancer are those whosefamily medical history indicates above average incidence of colorectal,stomach or esophageal cancer among family members and/or those who havealready developed colorectal, stomach or esophageal cancer and have beeneffectively treated who therefore face a risk of relapse and recurrence.

As used herein, the term “cancer characterized by expression of GCC” ismeant to refer to a cancer in which the cancer cells express GCC.Colorectal cells normally express GCC and continue to do so aftertransformation from normal to malignant. Accordingly, GCC is aneffective marker for identifying and targeting metastasized colorectalcancer. Likewise, GCC mRNA has been found in proximal tubule cells ofthe kidney, exocrine duct cells of the pancreas, submandibular glands,cells of the bile duct. suggesting that GCC may be expressed by thesecells. Esophageal and stomach cells normally do not express GCC but asthey develop into cancer, GCC begins to be expressed. Accordingly,detection of GCC at locations other than locations in which GCC isnormally expressed suggests cancer.

As used herein, the term “antisense composition” and “antisensemolecules” are used interchangeably and are meant to refer to compoundsthat regulate transcription or translation by hybridizing to DNA or RNAand inhibiting and/or preventing transcription or translation fromtaking place. Antisense molecules include nucleic acid molecules andderivatives and analogs thereof. Antisense molecules hybridize to DNA orRNA in the same manner as complementary nucleotide sequences doregardless of whether or not the antisense molecule is a nucleic acidmolecule or a derivative or analog. Antisense molecules may inhibit orprevent transcription or translation of genes whose expression is linkedto cancer.

Unless otherwise indicated, all chiral, diastereomeric and racemic formsare included in the present invention. Many geometric isomers ofolefins, C═N double bonds, and the like can also be present in thecompounds described herein, and all such stable isomers are contemplatedin the present invention. It will be appreciated that compounds of thepresent invention may contain asymmetrically substituted carbon atoms,and may be isolated in optically active or racemic forms. It is wellknown in the art how to prepare optically active forms, such as byresolution of racemic forms or by synthesis, from optically activestarting materials. All chiral, diastereomeric, racemic forms and allgeometric isomeric forms of a structure are intended, unless thespecific stereochemistry or isomer form is specifically indicated.

As used herein, the term “naturally occurring amino acids” means theL-isomers of the naturally occurring amino acids. The naturallyoccurring amino acids are glycine, alanine, valine, leucine, isoleucine,serine, methionine, threonine, phenylalanine, tyrosine, tryptophan,cysteine, proline, histidine, aspartic acid, asparagine, glutamic acid,glutamine, carboxyglutamic acid, arginine, ornithine and lysine. Unlessspecifically indicated, all amino acids referred to in this applicationare in the L-form.

As used herein, the term “side chain of a naturally occurring aminoacid” refers to the substituent on the a-carbon of an a amino acid. Thetem “polar side chain of a naturally occurring amino acid” refers to theside chain of a positively charged, negatively charged or hydrophilicamino acid. The tem “nonpolar side chain of a naturally occurring aminoacid” refers to the side chain of a hydrophobic amino acid. Side Chainsmay be independently selected from, and may independently terminate withreactive groups: H; C1-C10 alkyl; C2-C6 alkenyl; C3-C11 cycloalkyl;C4-C11 cycloalkylalkyl; C6-C10 aryl; C7-C11 arylalkyl; C2-C7alkylcarbonyl; C6-C10 arylcarbonyl; C2-C10 alkoxycarbonyl; C4-C11cycloalkoxycarbonyl; C7-C11bicycloalkoxycarbonyl; C6-C10aryloxycarbonyl; aryl(C1-C10 alkoxy)carbonyl; C1-C6alkylcarbonyloxy(C1-C4 alkoxy)carbonyl; C6-C10 arylcarbonyloxy(C1-C4alkoxy)carbonyl; C4-C11 cycloalkylcarbonyloxy(C1-C4 alkoxy)carbonyl;-(pyridyl)-; -(pyridazinyl)-; (pipirinyl)-;1,3-dioxa-cyclopenten-2-one-yl)methyloxy, C10 to C14;(5-aryl-1,3-dioxa-cyclopenten-2-one-yl)methylOxy; (R) (R)N—(C1-C10alkoxy)-; C3 to C11 cycloalkyl; C4 to C11 cycloalkylmethyl; C1-C6alkoxy; benzyloxy; C6 to C10 aryl, heteroaryl or heteroarylalkyl; C7 toC11 arylalkyl, adamantylmethyl or C1-C10 alkyl; azabicyclononyl,1-piperidinyl, 1-morpholinyl or 1-piperazinyl, each being optionallysubstituted with C1-C6 alkyl, C6-C10 aryl, heteroaryl, C7-C1, arylalkyl,C1-C6 alkylcarbonyl, C3-C7; cycloalkylcarbonyl, C1-C6 alkoxycarbonyl,C7-C11arylalkoxycarbonyl, C1-C6 alkylsulfonyl or C6-C10 arylsulfonyl;

As used herein a reactive group is an atom(s) which reacts selectivelywith another atom to form a covalent bond. Non-limiting examples are—(CH2)SH, —(CH2)nBr and —(CH2)C(═O)Cl.

As used herein, the term “positively charged amino acid” or “cationicamino acid” as used herein includes any naturally occurring or unnaturalamino acid having a positively charged side chain under normalphysiological conditions. Examples of positively charged naturallyoccurring amino acids are arginine, lysine and histidine.

As used herein, the term “negatively charged amino acid” includes anynaturally occurring or unnatural amino acid having a negatively chargedside chain under normal physiological conditions. Examples of negativelycharged naturally occurring amino acids are aspartic acid and glutamicacid.

As used herein, the term “hydrophilic amino acid” means any amino acidhaving an uncharged, polar side chain that is relatively soluble inwater. Examples of naturally occurring hydrophilic amino acids areserine, threonine, tyrosine, asparagine, glutamine, and cysteine.

As used herein, the term “hydrophobic amino acid” means any amino acidhaving an uncharged, nonpolar side chain that is relatively insoluble inwater. Examples of naturally occurring hydrophobic amino acids arealanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophanand methionine.

As used herein, the a “derivatized amino acid” is a native amino acidwhich has been chemically modified. Non-limiting examples arehydroxyl-proline, penicillamine, N-methyl-alanine.

As used herein, the amino acid mimetic (isostere) is an organic moleculewhich approximates the steric and electronic configuration of the aminoacid it is intended to replace. Non-limiting examples are, Amino AcidMimetic Cysteine 2-carboxy-3-thiopiperidine, penicillamine,homocysteine, 2-carboxy-3-bromo-pyridine, 2- bromo-benzoic acid,2-bromo-cyclohexanoic acid Proline Pipecolic acid, Oic,

As used herein, the term “beta turn” is defined as a 180 degree changein direction of a peptide chain in the span of 4 amino acid residues.The CO of the first residue (i) is hydrogen bonded to the NH of thefourth residue (i+3). In the case of STa, residue “i” is Asparagine11and residue i+3 is Cysteine14 Theory are many types of Beta turns. Theseturn types can be categorized by the phi and psi angles of the secondand third (i+1 and i+2 residues). The criteria for turn type I are givenin the table below: (i) (i + 1) (i + 2) (i + 3) residue residue residueresidue Turn Type phi angle psi angle phi angle psi angle I −60 −30 90 0I′ 60 30 90 0 II −60 120 80 0 II′ 60 −120 −80 0Of course, a turn found in an actual protein will not have the exactangles above, but if the angles are in proximity then the turn can beclassified accordingly.

STa has a type 1 beta turn from residues 11 through 14. An example ofanother type I turn is that which is found in the protein subtilisin.The turn consists of His39-Pro40-Asp41-Leu42. The CO of His39 (n) ishydrogen bonded to the NH of Leu42 (n+3), meeting the definition of aBeta turn. In addition the phi and psi angles for Pro40 (n+1) and Asp41(n+2) were measured using the Swiss PDB Viewer. These angle measurementsconfirm that this is a Type I turn. In addition, although difficult todiscern from the view shown here, the O₂ from the Proline extends to therear or “down” as is to be expected from a Type I Turn. (n + 1) Pro4.0(n + 1) Pro4.0 (n + 2) Asp4.1 (n + 2) Asp4.1 residue residue residueresidue psi angle psi angle psi angle psi angle −64.21 −17.19 −99.3212.92

In a Type I turn the hydrogen bond between CO of residue i and NH ofresidue i+3. The backbone dihedral angles of residue are (−60, −30) and(−90, 0) of residues i+1 and i+2, respectively of the type I turn.Proline is often found in position i+1 in type I turns as its phi angleis restricted to −60 and its imide nitrogen does not require a hydrogenbond. Glycine is favored in this position in the type II′ as it requiresa positive (left-handed) phi value.

For each type there are 4 sections for each residue. Each sectionrepresents a position in the turn. Within each section the first numberrepresents the number of examples, the second number is the potentialand the 3rd number is the significance as calculated by a d test. Valueswith mod(d)>=1.97 were taken as significant. position i position i + 1position i + 2 position i + 3 I 21 0.32 −5.73 40 0.60 −3.33 19 0.29−5.98 50 0.75 −2.07 F 40 0.81 −1.37 21 0.42 −4.13 56 1.13 0.95 50 1.010.08 V 33 0.39 −5.78 61 0.72 −2.61 42 0.50 −4.76 65 0.77 −2.16 L 68 0.68−3.38 70 0.70 −3.17 52 0.52 −5.05 74 0.74 −2.76 W 10 0.54 −1.98 12 0.65−1.51 24 1.30 1.31 21 1.14 0.60 M 16 0.65 −1.75 13 0.53 −2.36 10 0.41−2.97 15 0.61 −1.95 A 68 0.63 −3.99 115 1.07 0.76 86 0.80 −2.17 93 0.87−1.46 G 109 1.07 0.74 41 0.40 −6.29 62 0.61 −4.12 242 2.38 14.51 C 361.57 2.75 20 0.87 −0.62 22 0.96 −0.20 31 1.35 1.69 Y 30 0.66 −2.37 280.61 −2.67 41 0.90 −0.71 39 0.85 −1.02 P 76 1.31 2.40 203 3.49 19.46 120.21 −6.20 2 0.03 −7.54 T 81 1.11 0.95 69 0.94 −0.49 105 1.44 3.85 811.11 0.95 S 120 1.52 4.74 119 1.50 4.63 102 1.29 2.65 81 1.02 0.21 H 451.60 3.20 15 0.53 −2.52 28 0.99 −0.04 26 0.92 −0.42 E 54 0.74 −2.28 1121.54 4.72 88 1.21 1.83 74 1.02 0.13 N 125 2.25 9.50 38 0.68 −2.43 1262.26 9.63 60 1.08 0.59 Q 31 0.72 −1.91 35 0.81 −1.29 51 1.18 1.18 250.58 −2.84 D 180 2.51 13.15 86 1.20 1.72 182 2.54 13.40 80 1.11 0.99 K50 0.70 −2.57 85 1.20 1.70 72 1.01 0.11 78 1.10 0.85 R 36 0.64 −2.75 480.86 −1.11 49 0.88 −0.97 44 0.78 −1.65

From this, for STa, the preferred sequences for ST: Asp/Asn11-Pro12-Asp/Asn13-Gly/Cys14 compared to that seen in STa:Asn11-Pro12-Ala13-Cys14Asp>Asn>His>Cys>Ser>Pro>Thr>Gly

When compared to the ST analog Database, Asp, Asn, Val, His and Tyranalogs at residue 12 are provide potent agonists.Pro>>Glu>SerAsp>Lys>Ala

When compared to the ST analog Database, Pro, Aib, Val, Ala, Ile andD-Pro analogs at residue 12 all provide potent agonists.Asp>Asn>Thr>Trp>Ser>Glu>Gln>Lys

When compared to the ST analog Database, only Ala, Gly and Ser analogsat residue 13 provide potent agonists.Gly>Cys>Trp>Thr>Asp

When compared to the ST analog Database, no native substitutions arepossible at residue 12 which provide potent agonists. Moreover, only athioether crosslink (Hidaka) mutation exhibited any activity. There arenumerous non-native amino acids that form beta turns.

According to Sato (Biochem, 33: 8641-8650 (1994)), STa has a 310 helixfrom Cys⁶ through Cys⁹, with a characteristic H-bond between thecarbonyl oxygen of Cys⁶ and the amide nitrogen of Cys⁶. A 3₁₀-helix hasi,i+3H-bonds w/10 atoms between H-bonds.

As used herein the term “crosslinkers” is meant to refer to the bondsformed between functional groups on the two portions of the moleculelinked together by them, i.e the bonds that crosslink two parts of amolecule. The bonds formed by linkers include:

-   -   1. Disulfides [—S—S—]; thioethers [—S—]; thiosulfoxides        [—S(═O)—]; thiosulfonyl groups [—S(═O)₂—];    -   2. Amides, alkylamides and arylamides [—NR¹¹—C(═O)]; thioamides,        alkylthioamides and arylthioamides [—NR¹¹—C═S)—] wherein R¹¹ can        be —H, —OH, methylene groups, [—CR¹² ₂—]; methine groups,        [—CR¹²═CR¹²]; poly(methylene) groups, [—(CR¹² ₂)_(n)—] wherein n        is an integer from 2 to about 16 and can include linear and        branched saturated [—CR¹²═CR¹²—] and/or unsaturated [—CR¹²        ₂—CR¹² ₂—] groups. R¹² can be any atom, including hydrogen;    -   3. Oxycarbonyl groups [—O—C(═O)—]; aminocarbonyl groups        [—NH—C(═O)—]; carbonyl groups [—(C═O)—]; carbonyloxy groups        [—C(—O)—O—]; carbonate groups [—O—C(═O)—O—];        carbonyloxymethyleneoxycarbonylalkylene groups        [C(═O)—O—C(R²⁰R²¹)—O—C(═O)—(—CH2-)n—] where n′ is an integer        from 1 to 16 and each R²⁰ and R²¹ is independently selected from        the group consisting of H and methyl;    -   4. Urethane groups [—O—C(═O)—NH—]; thiourethane groups [—O—        (C═S)—NH—]    -   5. —NR³¹—CR³², R³³—CR³⁴R³⁵— wherein R³¹ can be selected from the        group consisting of H, an alkyl group of from 1 to about 16        carbon atoms which may be linear, branched, saturated,        unsaturated, or contain a carbocyclic ring of from 3 to about 10        carbon atoms, or a carbonylalkyl group wherein the alkyl group        is defined immediately above; R³² and R³³ are independently        selected from the group consisting of H, an alkylene group of        from 1 to about 16 carbon atoms, which may be linear, branched,        saturated or unsaturated, and can contain a carbocyclic ring of        from 3 to about 10 carbon atoms R³⁴ and R³⁵ are independently        selected from the group consisting of H, an alkyl group of from        1 to about 16 carbon atoms which may be linear, branched,        saturated, unsaturated, or contain a carbocyclic ring of from 3        to about 10 carbon atoms, or a carbonylalkyl group wherein the        alkyl group is defined above, or preferably, where both R³⁴ and        R³⁵ are taken together form a carbonyl group; and wherein at        least one of R³²R³³ is not H, preferably —NR³¹—CR³²,        R³³—CR³⁴R³⁵— are selected from the group consisting of native        and non-native amino acids;    -   6. Methylene groups, [—CR⁴¹ ₂—); methine groups, [—CR⁴¹═CR⁴¹];        poly(methylene) groups, [—(CR⁴¹ ₂)n—] wherein n is an integer        from 2 to about 16 and can include linear and branched saturated        [—CR⁴¹═CR⁴¹—] and/or unsaturated [—CR⁴¹ ₂—CR⁴¹ ₂—) groups. R⁴¹        can be any atom, including hydrogen;    -   7. Aryl- and alkylenecarbonyl groups[—(CH₂)_(n)—C(═O)-] wherein        n is an integer from 1 to about 16;    -   8. Ethylenesulfonylethylene groups [—CH₂CH₂—S(═O)(═O)₂CH₂CH₂]    -   9. Ethylenesulfonylmethyleneoxymethylenesulfonylethylene groups        [—CH₂ CH₂—S(═O)₂—CH₂—O—CH₂—S(═O)₂—CH₂CH₂—];    -   10. Ethylenesulfonylmethylenesulfonylethylene groups        CH₂CH₂—S(═O)₂—CH₂—S(═O)₂—CH₂CH₂—];    -   11. Carbonyloxy groups [—C(═O)—O—];    -   12. Aryl- and alkylenecarbonyloxymethyleneoxycarbonylalkylene        groups [—(—CH₂—)_(n), C(═O)—O—C(R¹⁵R⁵²)—O—C(═O)—(—CH₂—)n-] where        each n′ is independently selected from the group of integers        from 1 to 16 and each R⁵¹ and R⁵² is independently selected from        the group consisting of H and methyl;    -   13. Carbonylalkylenecarbonyl groups [—C(═O)—(CH₂)_(w)—C(═O)—]        wherein w is an integer from 1 to about 6, such as succinate and        adipate.

As used herein, guanylyl cyclase C-mediated diarrhea or GC-C-mediateddiarrhea is meant to refer to diarrhea which is associated with guanylylcyclase C (GC-C) enzyme activity. An example of GC-C-mediated diarrheais infectious diarrhea which is brought about by GC-C enzyme activationwhen the E. coli heat stable toxin (ST) binds to receptors on cellswithin the colon of an individual. Infectious diarrhea is also referredto herein as E. coli heat stable toxin induced diarrhea.

As used herein “alkyl” is meant to “Alkyl” groups as well as unsaturatedand substituted forms such as “Substituted Alkyl,” “Alkenyl,”“Substituted Alkenyl,” “Alkynyl,” “Substituted Alkynyl” and “Alkoxy.”

“Alkyl:” refers to a saturated branched, straight chain or cyclichydrocarbon group. Typical alkyl groups include, but are not limited to,methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, pentyl,isopentyl, hexyl, and the like. In preferred embodiments, the alkylgroups are (C₁-C₆) alkyl, with (C₁-C₃) being particularly preferred.

“Substituted Alkyl:” refers to an alkyl group wherein one or morehydrogen atoms are each independently replaced with other substituents.

“Alkenyl:” refers to an unsaturated branched, straight chain or cyclichydrocarbon group having at least one carbon-carbon double bond. Thegroup may be in either the cis or trans conformation about the doublebond(s). Typical alkenyl groups include, but are not limited to,ethenyl, propenyl, isopropenyl, butenyl, isobutenyl, tert-butenyl,pentenyl, hexenyl and the like. In preferred embodiments, the alkenylgroup is a (C₁-C₆) alkenyl, with (C₁-C₃) being particularly preferred.

“Substituted Alkenyl:” refers to an alkenyl group wherein one or morehydrogen atoms are each independently replaced with other substituents.

“Alkynyl:” refers to an unsaturated branched, straight chain or cyclichydrocarbon group having at least one carbon-carbon triple bond. Typicalalkynyl groups include, but are not limited to, ethynyl, propynyl,butynyl, isobutynyl, pentynyl, hexynyl and the like. In preferredembodiments, the alkynyl group is (C₁-C₆) alkynyl, with (C₁-C₃) beingparticularly preferred.

“Substituted Alkynyl:” refers to an alkynyl group wherein one or morehydrogen atoms are each independently replaced with other substituents.

“Alkoxy:” refers to an —OR group, where R is alkyl, alkenyl or alkynyl,as defined above.

As used herein “aryl” is meant to “Aromatic moiety,” “SubstitutedAromatic Moiety,” “Heteroaromatic moiety” and “SubstitutedHeteroaromatic moiety.”

“Aromatic moiety:” refers to a moiety having an unsaturated cyclichydrocarbon group which has a conjugated (4n+2).pi. electron system.Typical aromatic moieties include, but are not limited to, benzene,naphthalene, anthracene, azulene, indacene, and the like. In preferredembodiments, the aromatic moiety contains 5-20 carbons in the ringsystem, with 5-10 carbon atoms being particularly preferred.

“Substituted Aromatic Moiety:” refers to an aromatic moiety wherein oneor more hydrogen atoms are each independently replaced with othersubstituents.

“Heteroaromatic moiety:” refers to an aromatic moiety wherein one ormore of the ring carbon atoms is replaced with another atom such as N, Oor S. Typical heteroaromatic moieties include, but are not limited to,pyran, pyrazole, pyridine, pyrrole, pyrazine, pyridazine, pyrimidine,pyrrolizine, quinazoline, quinoline, quinolizine, quinoxaline,selenophene, thiophere, tellurophene, xanthene and the like.

“Substituted Heteroaromatic moiety:” refers to a heteroaromatic moietywherein one or more hydrogen atoms are each independently replaced withother substituents.

Overview

The present invention arises from the discovery that identification ofstructural determinants of GCC ligands necessary for GCC binding and GCCactivation. Accordingly, retention of the structural determinantsrequired for GCC binding and GCC activation yields compounds which areGCC agonists, i.e. compounds that bind to GCC and activate it. Compoundsof the invention include agonists which induce cyclic GMP accumulationassociated with the binding of GCC to natural ligands. On the otherhand, retention of the structural determinants required for GCC bindingand elimination of structural determinants required for GCC activationyields compounds which are GCC compound of the inventions, i.e.compounds that bind to GCC without activating it. Compounds of theinvention include compound of the inventions that bind to GCC but do notinduce cyclic GMP accumulation associated with the binding of GCC tonatural ligands.

Compounds of the invention that are agonists have several uses includingas anti-cancer compounds. They may be used alone or in combination withother therapeutic compounds. Further, they may be conjugated orunconjugated. The present invention relates to methods of treatingindividuals who have or are suspected of having cancer by administeringcompounds of the invention. Such compounds are useful in methods ofinhibiting metastasis.

Compounds of the invention that are compound of the inventions haveseveral uses including as anti-diarrhea compounds. They may be usedalone or in combination with other therapeutic compounds. Further, thepresent invention relates to methods of treating individuals who have orare suspected of being at an elevated risk of contracting diarrhea byadministering compounds of the invention.

Because compounds of the invention bind to GCC, the peptides and peptideanalogues of the invention can be used to prepare imaging agents,therapeutics and diagnostic reagents related to the detection andtreatment of cancer characterized by cells expressing GCC. As notedabove, agonist peptides and peptide analogues of the invention can beused unconjugated to activate GCC in methods of treating cancer andpreventing metastasis. Compound of the invention peptides and peptideanalogues of the invention can be used to inhibit GCC activation inmethods of treating diseases and disorders in which GCC activation hasundesirable clinical effects which can be reduced or inhibited byinhibiting GCC activation.

Natural ligands of GCC include endogenous natural ligands guanylin anduroguanylin and the heat-stable enterotoxins (STs), a family ofstructurally-related peptides produced by different organisms including,but not limited to, E. coli, Yersinia, Enterobacter, and Vibrio. Table Ishows the amino acid sequences of these natural ligands. The numericalpositions referred to in the Table are the positions from the widelystudied peptide E. coli. STa. TABLE 1 Position 5 6 9 10 14 17 E. coliSTa N T F Y C C E L C C N P A C A G C Y E. coli STh S S S N Y C C E L CC N P A C T G C Y Yersinia S S D W D Y C C D L C C N P A C A G C YVibrio I D C C E I C C N P A C F G C L N Guanylin P N T C E I C A Y A AC T G C Uroguanylin Q E D C E I C I N V A C T G CThe ST peptides each have six cysteine residues while the endogenousligands have four each. In the ST peptides, the six cysteines form threedi-sulphide bonds: amino acid 5 forms a disulfide bond with amino acid10 (Loop A); amino acid 6 forms a disulfide bond with amino acid 14(Loop B); and amino acid 8 forms a disulfide bond with amino acid 17(Loop C). In the endogenous peptides, the four cysteines form twodi-sulphide bonds: amino acid 6 forms a disulfide bond with amino acid14 (Loop B), and amino acid 8 forms a disulfide bond with amino acid 17(Loop C). In each peptide a dipeptide occurs between position 6 and 9which produces a 3₁₀-helix turn. Each peptide includes a Type II (β)turn between positions 9 and 14.

Some aspects of the present invention provides GCC agonists which retainthe 3₁₀-helix turn between positions 6 and 9 and the β turn betweenpositions 9 and 14 while retaining the crosslinking between position 5and 10 (Loop A), while eliminating the crosslinking between position 6and 14 (Loop B) and the crosslinkage between positions 9 and 17 (LoopC). These compounds are referred to as Loop A compound of theinventions. It has been discovered that the retention of the threestructural determinants (3₁₀-helix turn between 6 and 9, β turn between9 and 14 and 5-10 cross linkage) and elimination of the two structuraldeterminants (6-14 cross linkage and 9-17 cross linkage) yieldscompounds which bind to GCC but do not activate the signal cascadeassociated with GCC binding to natural ligands. In some embodiments, theamino acid at position 5 can be deleted in which case the amino acid atposition 6 binds to the amino acid at 10. In some embodiments, the aminoacid at position 10 can be deleted in which case the amino acid atposition 5 binds to the amino acid at 9. In some embodiments, the aminoacid at position 5 and at position 10 can be deleted in which case theamino acid at position 6 binds to the amino acid at 9. In someembodiments, the amino acid at position 5 can be present but does notcross link and the amino acid at position 6 crosslinks to the amino acidposition 10. In some embodiments, the amino acids at position 5 and atposition 10 can be present but does not cross link and the amino acid atposition 6 crosslinks to the amino acid position 9. In some embodiments,the amino acid at position 10 can be present but does not cross link andthe amino acid at position 5 crosslinks to the amino acid position 9.Amino acids or other moieties may be linked to amino acid 14. In someembodiments, the position at the C terminus of amino acid 14 is COOH. Insome embodiments, the position at the C terminus of amino acid 14 is1-30 amino acids, such as for example native sequences 15-18 providedthe amino acid at position 17 does not cross link.

Some aspects of the present invention provides GCC agonists which retainthe 3₁₀-helix turn between positions 6 and 9 and the β turn betweenpositions 9 and 14 while retaining the crosslinking between position 6and 14 (Loop B), eliminating the crosslinking between position 9 and 17(Loop C) and optionally retaining the crosslinkage between positions 5and 10 (Loop A). If the compound does not have a tail linked to the Cterminal of position 14 the compound is a Loop B agonists. In someembodiments, the compound has such a tail but it is degradable by aenzyme or other means present in the individual to whom it isadministered. In such cases, the compound is an “in vivo agonist” inthat it will act as a compound of the invention in vitro but will beconverted to an agonist in vivo. An in vivo agonist can be maintained asa compound of the invention in vivo if the enzyme which degrades itstail is inhibited. For example, an in vivo Loop B agonist with a tailthat is removed by elastase, such as AGA, can be maintained as a Loop Bcompound of the invention in vivo by administering an elastase inhibitorin conjunction with it. In some embodiments, the Loop B compound of theinvention serves as a prodrug which is converted to a Loop B agonist invivo by a means already present in the individual or by the addition ofan agent that will convert it. It has been discovered that the retentionof the three structural determinants (3₁₀-helix turn between 6 and 9, Pturn between 9 and 14 and 6-14 cross linkage) and elimination of thestructural determinant (9-17 cross linkage) yields compounds which bindto GCC and, provided they have no tail at the C terminus, activate thesignal cascade associated with GCC binding to natural ligands. In someembodiments, the amino acid at position 5 can be deleted. In someembodiments, the amino acid at position 10 can be deleted. In someembodiments, the amino acids at positions 5 and 10 can be deleted. Insome embodiments, the amino acids at position 5 and 10 are present butdo not crosslink. In some embodiments, the amino acids at position 5 and10 are present and crosslinked to each other. Amino acids or othermoieties may be linked to amino acid 14 provided such amino acids orother moieties can be cleaved or otherwise removed form the molecule invivo if the compound is to be an agonist. In some embodiments, theposition at the C terminus of amino acid 14 is COOH. In someembodiments, the position at the C terminus of amino acid 14 is 1-30amino acids, such as for example native sequences 15-18 provided theamino acid at position 17 does not cross link.

Compounds of the Invention

The compounds of the invention have structures according toR1-R2-R3-R4-R5-R6-R7  (I)wherein:

-   -   R1 is combination of 1-50 natural amino acids, 1-50 blocked        amino acids, 1-50 synthetic amino acids, 1-50 derivatized amino        acids; 1-50 amino acid mimetics, a 1-50 alkyls, one or more 1-50        substituted alkyls, one or more 1-50 aryls, one or more 1-50        substituted aryls, one or more 1-50 alkylaryls, one or more 1-50        substituted alkylaryls or combinations thereof;    -   R2 is a natural amino acid, blocked amino acid, synthetic amino        acid, derivatized amino acid; an amino acid mimetic which        optionally crosslinks, as defined herein, with R6;    -   R3 is a natural amino acid, blocked amino acid, synthetic amino        acid, derivatized amino acid; an amino acid mimetic provided it        crosslinks, as defined herein, with R8;    -   R4 is dipeptide comprising one or two natural amino acids, one        or two blocked amino acids, one or two synthetic amino acids,        one or two derivatized amino acids; one or two amino acid        mimetics, or a dipeptide mimetic, wherein said dipeptide or        dipeptide mimetic forms a 3 ₁₀-helix turn group linking R3 to R5    -   R5 is a natural amino acid, blocked amino acid, synthetic amino        acid, derivatized ammo acid; an amino acid mimetic provided it        does not crosslink with R9    -   R6 is a natural amino acid, blocked amino acid, synthetic amino        acid, derivatized amino acid; an amino acid mimetic which        optionally crosslinks, as defined herein, with R2;    -   R7 is tripeptide comprising one, two or three natural amino        acids, one, two or three blocked amino acids, one, two or three        amino acid mimetics, or a tripeptide mimetic, wherein said        tripeptide or tripeptide mimetic forms a beta turn group linking        R6 to R8    -   R8 is a natural amino acid, blocked amino acid, synthetic amino        acid, derivatized amino acid; an amino acid mimetic provided it        crosslinks, as defined herein, with R3;    -   R9 is a combination of 1-50 natural amino acids, 1-50 blocked        amino acids, 1-50 synthetic amino acids, 1-50 derivatized amino        acids; 1-50 amino acid mimetics, a 1-50 alkyls, one or more 1-50        substituted alkyls, one or more 1-50 aryls, one or more 1-50        substituted aryls, one or more 1-50 alkylaryls, one or more 1-50        substituted alkylaryls or combinations thereof provided that it        does not crosslink to R5.

According to aspects of the present invention, compounds of theinvention have the structure of formula (I) provided that the compoundsis not:C-hC-E-L-A-C-N-P-A-X; hCC-E-L-A-X-N-P-A-C;C-C-E-L-A-C-N-P-A-C;C-C-E-L-A-C-N-P-A-C-T-G-A;Y-Ca-C-E-L-F-Ca-N-P-A-C;Y-C-C-E-L-mA-C-N-P-A-C;Y-C-C-E-mL-A-C-N-P-A-C;C-C-E-L-Cm-C-N-P-A-C-A-G-Cm;C-C-E-L-Ca-C-N-P-A-C-A-G-Ca; andX-Ca-E-L-A-hC-N-P-A-Ca;wherein

-   hC is Homocysteine;-   X is Alanine linked to hC via a covalent CH2-S bond between the Ala    side chain and the hC side chain;-   Cm is Cysteine(S-methoxybenzyl); and-   Ca is Cysteine(S-acetamidomethyl).

In some preferred embodiments, R1 is preferably 1-50 natural aminoacids, 1-50 blocked amino acids, 1-50 synthetic amino acids, 1-50derivatized amino acids; 1-50 amino acid mimetics, a 1-50 alkyls, one ormore 1-50 substituted alkyls, one or more 1-50 aryls, one or more 1-50substituted aryls, one or more 1-50 alkylaryls, one or more 1-50substituted alkylaryls or combinations thereof. In some embodiments, R1is 0-20 amino acids. In some embodiments R1 is smaller than 1000daltons. In some embodiments, R1 is Y, NTFY, SSSNY, SSDWDY, ID, PN, orQE.

In some preferred embodiments, R2 is a Cysteine side chain analog[—(CR2)n-S, or —(CR2)n-H; where n is C1-C10, R is any atom and H is areactive group], or aryl, alkyl and arylalkyl groups that may or may notcrosslink to Cys10. In some embodiments, R2 is2-carboxy-3-bromo-pyridine, 2-bromo-benzoic acid, 2-bromo-cyclohexanoicacid. R2 is preferably C cross linked to R6, blocked C such as Cmob,cross linked to R6 by a Carba link, any non-crosslinking aa or mimetic.

In some preferred embodiments, R3 is a Cysteine side chain analog[—(CR2)n-S, or —(CR2) n-H; where n is C1-C10, R is any atom and H is areactive group], an alpha, beta or gamma amino acid capable of forming acrosslink with Cys14 via a side chain functional group (see side chains,above), an amino acid mimetic, such as 2-carboxy-3-thiopiperidine, whichconstrain the dihedral angles or the side chain to favorableconfigurations. R3 is preferably C crosslinked to C at R8 or any othermoiety crosslinked to R8.

In some preferred embodiments, R4 is a single amino acid (all types),dipeptide, dipeptide mimetic and chain links, particularly structureswith 4-8 atoms bridging the gap between R3 and R5, most preferablystructures with 6 atoms bridging the gap between R3 and R5. Preferredamino acids for R4 are sarcosine, ornithine and lysine. Glu7 preferredamino acid replacements are Gln, Gly, Ser, Ala, Pro, Asp, Asn andL-octahydroindole-2-carboxyilic acid, and their N-alkyl, N-hydroxyl, orN-aryl analogs. Leu8 preferred amino acid replacements are Ile,Norleucine, Met, Val, Phe, Trp, Tyr, Gin, Gly, Ala, Pro andL-1,2,3,4-tetrahydroisoquinoline-3-carboxyilic acid, and their N-alkyl,N-hydroxyl, or N-aryl analogs. R4 is preferably a dipeptide such as E-L,or E-NmethylLeu.

In some preferred embodiments, R5 is a single amino acid (all types),preferably hydrophobic or neutral ones, and their N-alkyl, N-hydroxyl,or N-aryl analogs, more preferably Ala, N(Me)-Ala, Thr, Leu, N(Me)-Leu,Phe and blocked Cysteine, Serine and Threonine, an amino acid mimetic,such as L-1,2,3,4-tetrahydroisoquinoline-3-carboxyilic acid, whichconstrain the dihedral angles or the side chain to favorableconfigurations. In some preferred embodiments, R5 may be comprised ofamino acids or organic molecules with a hydrophobicity of at least −2.0(Glutamic acid is the lowest native AA with a value of −1.22), andpreferably a value greater than zero; and more preferably a valuegreater than one. Hydrophobicity is determined by the method of Sweetand Eisenberg (Sweet R. M., Eisenberg D., J. Mol. Biol.171:479-488(1983) which is incorporated herein by reference). R5 ispreferably A L, T, F, V, I, Y, NmethylLeu, NmethylAlam, and NmethylVal.

In some preferred embodiments, R6 is a single amino acid (all types), oran N-alkyl, N-hydroxyl, or N-aryl analog of one, an amino acid mimetic,such as which constrain the dihedral angles or the side chain tofavorable configurations. R6 is preferably C cross linked to R2, blockedC such as Cmob, cross linked to R2 by a Carba link any non-crosslinkingamino acid or mimetic.

In some preferred embodiments, R7 is a dipeptide mimetic, such asbicycle-L-seryl-proline, Btd, APM, ACTB, or ACDN

In some embodiments, R7 is X-Y-Z wherein

X is a single amino acid (all types), preferably Asn, Asp, Ala, His,GlnGly, beta-Alanine), or a short one (Ala, Ser, Thr, Asp), and theirN-alkyl, N-hydroxyl, or N-aryl analogs or a synthetic amino acids,preferably Isoasparagine or beta alanine.

Y is a single amino acids (all types), preferably Pro, Ile, Ala, Val(both D and L) and their N-alkyl, N-hydroxyl, or N-aryl analogs.Synthetic amino acids, such as homopro, Nipecotic acid, isonipecoticacid, Oic, Tic, Aib, aminobenzoate, carboxypiperidine, azetidinecarboxylate and aminocyclopentene carboxylic acid. Amino acid mimetics,such as L-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, whichconstrain the dihedral angles or the side chain to favorableconfigurations.

Z is a single amino acid (all types), preferably with no sidechain (Gly,beta-Alanine), or a short one (Ala, Ser, Thr, Asp), and their N-alkyl,N-hydroxyl, or N-aryl analogs, more preferably Ala, N(Me)-Ala, and Gly.

R7 is preferably a tripeptide N-P-A.

In some preferred embodiments, R8 is a Cysteine side chain analog[—(CR2)n-S, or —(CR2)n-H; where n is C1-C10, R is any atom and H is areactive group], an alpha, beta or gamma amino acids capable of forminga crosslink with Cys 14 via a side chain functional group (see sidechains, above), an amino acid mimetic, such as2-carboxy-3-thiopiperidine, which constrain the dihedral angles or theside chain to favorable configurations. R8 is preferably C crosslinkedto C at R3 or any other moiety crosslinked to R3.

In some preferred embodiments, R9 is preferably smaller than 1000daltons. R9 is preferably 0-20 amino acids, AGA or Y.

It is preferred that the compound of the invention be as small aspossible. Thus it is preferred that the compound of the invention be anon-peptide small molecule or small peptide, preferably less than 25amino acids, more preferably less than 20 amino acids. In someembodiments, the GCC compound of the invention binding is in the form ofa conjugated composition is less than 15 amino acids. The compound ofthe invention comprising 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 aminoacids may be used according to the present invention. It is within thescope of the present invention to include larger molecules which serveas compounds of the invention.

The present invention also relates to compounds that are Loop A compoundof the inventions. The present invention relates to compounds havingFormula (II):R201-R202-R203-R204-R205-R206-R207 wherein:  (II)

-   -   R201 is absent or 1-50 natural amino acids, 1-50 blocked amino        acids, 1-50 synthetic amino acids, 1-50 derivatized amino acids;        1-50 amino acid mimetics, a 1-50 alkyls, one or more 1-50        substituted alkyls, one or more 1-50 aryls, one or more 1-50        substituted aryls, one or more 1-50 alkylaryls, one or more 1-50        substituted alkylaryls or combinations thereof;    -   R202 is absent, or is a natural amino acid, blocked amino acid,        synthetic amino acid, derivatized amino acid or an amino acid        mimetic which does not crosslink to any other amino acid, or is        a natural amino acid, blocked amino acid, synthetic amino acid,        derivatized amino acid or amino acid mimetic that crosslinks        with R205 or R206, wherein if R202 is absent or a natural amino        acid, blocked amino acid, synthetic amino acid, derivatized        amino acid or an amino acid mimetic which does not crosslink to        any other amino acid then R203 is a natural amino acid, blocked        amino acid, synthetic amino acid, derivatized amino acid or        amino acid mimetic that crosslinks with R205 or R206;    -   R203 is absent, or is a natural amino acid, blocked amino acid,        synthetic amino acid, derivatized amino acid or an amino acid        mimetic which does not crosslink to any other amino acid, or if        R202 is absent or is a natural amino acid, blocked amino acid,        synthetic amino acid, derivatized amino acid or an amino acid        mimetic which does not crosslink to any other amino acid then        R203 is a natural amino acid, blocked amino acid, synthetic        amino acid, derivatized amino acid or amino acid mimetic that        crosslinks with R205 or R206;    -   R204 is one or two natural amino acids, one or two blocked amino        acids, one or two synthetic amino acids, one or two derivatized        amino acids, one or two amino acid mimetics, or a dipeptide        mimetic or combinations thereof, wherein R204 forms a 3₁₀-helix        turn group linking R203 to R205;    -   R205 is absent, or is a natural amino acid, blocked amino acid,        synthetic amino acid, derivatized amino acid or an amino acid        mimetic which does not crosslink to any other amino acid, or if        R206 is absent or is a natural amino acid, blocked amino acid,        synthetic amino acid, derivatized amino acid or an amino acid        mimetic which does not crosslink to any other amino acid then        R205 is a natural amino acid, blocked amino acid, synthetic        amino acid, derivatized amino acid or amino acid mimetic that        crosslinks with R202 or R203;    -   R206 is absent, or is a natural amino acid, blocked amino acid,        synthetic amino acid, derivatized amino acid or an amino acid        mimetic which does not crosslink to any other amino acid, or is        a natural amino acid, blocked amino acid, synthetic amino acid,        derivatized amino acid or amino acid mimetic that crosslinks        with R202 or R203, wherein if R206 is absent or a natural amino        acid, blocked amino acid, synthetic amino acid, derivatized        amino acid or an amino acid mimetic which does not crosslink to        any other amino acid then R205 is a natural amino acid, blocked        amino acid, synthetic amino acid, derivatized amino acid or        amino acid mimetic that crosslinks with R202 or R203; and    -   R207 is absent or 1-50 natural amino acids, 1-50 blocked amino        acids, 1-50 synthetic amino acids, 1-50 derivatized amino acids;        1-50 amino acid mimetics, a 1-50 alkyls, one or more 1-50        substituted alkyls, one or more 1-50 aryls, one or more 1-50        substituted aryls, one or more 1-50 alkylaryls, one or more 1-50        substituted alkylaryls or combinations thereof.

In some preferred embodiments, R201 is preferably 1-50 natural aminoacids, 1-50 blocked amino acids, 1-50 synthetic amino acids, 1-50derivatized amino acids; 1-50 amino acid mimetics, a 1-50 alkyls, one ormore 1-50 substituted alkyls, one or more 1-50 aryls, one or more 1-50substituted aryls, one or more 1-50 alkylaryls, one or more 1-50substituted alkylaryls or combinations thereof. In some embodiments, R1is 0-20 amino acids. In some embodiments R201 is smaller than 1000daltons. In some embodiments, R201 is Y, NTFY, SSSNY, SSDWDY, ID, PN, orQE.

In some preferred embodiments, R202 is a Cysteine. In some preferredembodiments, R202 is a penacillamine. In some preferred embodiments,R202 is a Cysteine side chain analog [—(CR2)n-S, or —(CR2)n-H; where nis C1-C10, R is any atom and H is a reactive group], or aryl, alkyl andarylalkyl groups that may or may not crosslink to Cys10. In someembodiments, R202 is 2-carboxy-3-bromo-pyridine, 2-bromo-benzoic acid,2-bromo-cyclohexanoic acid. R202 is preferably C cross linked to R206,or R205, or blocked C such as Cmob, cross linked to R206 by a Carbalink, any non-crosslinking aa or mimetic.

In some preferred embodiments, R203 is absent or cannot form a crosslink(such as y for example being a blocked Cys, Alanine, Serrine or otheralpha amino acid). In embodiments in which R202 does not form acrosslink, R203 forms a crosslink and may be for example Cysteine,penacillamine or another Cysteine side chain analog [—(CR2)n-S, or—(CR2)n-H; where n is C1-C10, R is any atom and H is a reactive group],or aryl, alkyl and arylalkyl groups. In some such embodiments, R203 maybe as R202 described above and cross link with R206 or R205.

In some preferred embodiments, R204 is a single amino acid (all types),dipeptide, dipeptide mimetic and chain links, particularly structureswith 4-8 atoms bridging the gap between R203 and R205, most preferablystructures with 6 atoms bridging the gap between R203 and R205.Preferred amino acids for R204 are sarcosine, ornithine and lysine. Glu7preferred amino acid replacements are Gln, Gly, Ser, Ala, Pro, Asp, Asnand L-octahydroindole-2-carboxyilic acid, and their N-alkyl, N-hydroxyl,or N-aryl analogs. Leu8 preferred amino acid replacements are Ile,Norleucine, Met, Val, Phe, Trp, Tyr, Gln, Gly, Ala, Pro andL-1,2,3,4-tetrahydroisoquinoline-3-carboxyilic acid, and their N-alkyl,N-hydroxyl, or N-aryl analogs. R204 is preferably a dipeptide such asE-L, or E-NmethylLeu.

In some preferred embodiments, R205 is a single amino acid (all types),preferably hydrophobic or neutral ones, and their N-alkyl, N-hydroxyl,or N-aryl analogs, more preferably Ala, N(Me)-Ala, Thr, Leu, N(Me)-Leu,Phe and blocked Cysteine, Serine and Threonine, an amino acid mimetic,such as L-1,2,3,4-tetrahydroisoquinoline-3-carboxyilic acid, whichconstrain the dihedral angles or the side chain to favorableconfigurations. In some preferred embodiments, R205 may be comprised ofamino acids or organic molecules with a hydrophobicity of at least −2.0(Glutamic acid is the lowest native AA with a value of −1.22), andpreferably a value greater than zero; and more preferably a valuegreater than one. Hydrophobicity is determined by the method of Sweetand Eisenberg (Sweet R. M., Eisenberg D., J. Mol. Biol.171:479-488(1983) which is incorporated herein by reference). R205 ispreferably A L, T, F, V, I, Y, NmethylLeu, NmethylAlam, and NmethylVal.In embodiments, R205 is absent or cannot form a crosslink (such as y forexample being a blocked Cys, Alanine, Serrine or other alpha aminoacid). In embodiments in which R206 does not form a crosslink, R205forms a crosslink and may be for example Cysteine, penacillamine oranother Cysteine side chain analog [—(CR2)n-S, or —(CR2)n-H; where n isC1-C10, R is any atom and H is a reactive group], or aryl, alkyl andarylalkyl groups. In some such embodiments, R205 may be as R202described above and cross link with R202 or R203.

In some preferred embodiments, R206 is a Cysteine. In some preferredembodiments, R206 is a penacillamine. In some preferred embodiments,R206 is a Cysteine side chain analog [—(CR2)n-S, or —(CR2)n-H; where nis C1-C10, R is any atom and H is a reactive group], or aryl, alkyl andarylalkyl groups. In some embodiments, R206 is2-carboxy-3-bromo-pyridine, 2-bromo-benzoic acid, 2-bromo-cyclohexanoicacid. R206 is preferably C cross linked to R202, or R202, or blocked Csuch as Cmob, cross linked to R202 by a Carba link, any non-crosslinkingaa or mimetic.

In some preferred embodiments, R207 is absent, COOH, NPAAAGCacmY, orST11-18 with a change at 17 to remove crosslinking function.

The present invention also relates to compounds that are Loop Bcompounds. The present invention relates to compounds having Formula(III):R301-R302-R303-R304-R305-R306-R307-R308-R309  (III)wherein:

-   -   R301 is combination of 1-50 natural amino acids, 1-50 blocked        amino acids, 1-50 synthetic amino acids, 1-50 derivatized amino        acids; 1-50 amino acid mimetics, a 1-50 alkyls, one or more 1-50        substituted alkyls, one or more 1-50 aryls, one or more 1-50        substituted aryls, one or more 1-50 alkylaryls, one or more 1-50        substituted alkylaryls or combinations thereof;    -   R302 is a natural amino acid, blocked amino acid, synthetic        amino acid, derivatized amino acid, or an amino acid mimetic        wherein R302 may crosslink with R306;    -   R303 is a natural amino acid, blocked amino acid, synthetic        amino acid, derivatized amino acid or amino acid mimetic that        crosslinks with R308;    -   R304 is one or two natural amino acids, one or two blocked amino        acids, one or two synthetic amino acids, one or two derivatized        amino acids, one or two amino acid mimetics, or a dipeptide        mimetic or combinations thereof, wherein R304 forms a ³10-helix        turn group linking R303 to R305;    -   R305 is a natural amino acid, blocked amino acid, synthetic        amino acid, derivatized amino acid or amino acid mimetic that        does not crosslink with R309    -   R306 is a natural amino acid, blocked amino acid, synthetic        amino acid, derivatized amino acid, or amino acid mimetic        wherein R306 may crosslink with R302;    -   R307 is one, two or three natural amino acids, one, two or three        blocked amino acids, one, two or three amino acid mimetics, or a        tripeptide mimetic or combinations thereof, wherein R307 forms a        beta turn group linking R306 to R308    -   R308 is a natural amino acid, blocked amino acid, synthetic        amino acid, derivatized amino acid or amino acid mimetic that        crosslinks with R303;    -   R309 is absent or 1-50 natural amino acids, 1-50 blocked amino        acids, 1-50 synthetic amino acids, 1-50 derivatized amino acids;        1-50 amino acid mimetics, a 1-50 alkyls, one or more 1-50        substituted alkyls, one or more 1-50 aryls, one or more 1-50        substituted aryls, one or more 1-50 alkylaryls, one or more 1-50        substituted alkylaryls or combinations thereof that does not        crosslink to R305;    -   wherein the structure of the compounds is not:        C-hC-E-L-A-C-N-P-A-X; hC        C-E-L-A-X-N-P-A-C;        C-C-E-L-A-C-N-P-A-C;        C-C-E-L-A-C-N-P-A-C-T-G-A;        Y-Ca-C-E-L-F-Ca-N-P-A-C;        Y-C-C-E-L-mA-C-N-P-A-C;        Y-C-C-E-mL-A-C-N-P-A-C;        C-C-E-L-Cm-C-N-P-A-C-A-G-Cm;        C-C-E-L-Ca-C-N-P-A-C-A-G-Ca; or        X-Ca-E-L-A-hC-N-P-A-Ca;        wherein

-   hC is Homocysteine;

-   X is Alanine linked to hC via a covalent CH2-S bond between an Ala    side chain and an hC side chain;

-   Cm is Cysteine(S-methoxybenzyl); and

-   Ca is Cysteine(S-acetamidomethyl).

In some preferred embodiments, R301 is preferably 1-50 natural aminoacids, 1-50 blocked amino acids, 1-50 synthetic amino acids, 1-50derivatized amino acids; 1-50 amino acid mimetics, a 1-50 alkyls, one ormore 1-50 substituted alkyls, one or more 1-50 aryls, one or more 1-50substituted aryls, one or more 1-50 alkylaryls, one or more 1-50substituted alkylaryls or combinations thereof. In some embodiments,R301 is 0-20 amino acids. In some embodiments R301 is smaller than 1000daltons. In some embodiments, R301 is Y, NTFY, SSSNY, SSDWDY, ID, PN, orQE.

In some preferred embodiments, R302 is a Cysteine side chain analog[—(CR2)n--S, or —(CR2)n-H; where n is C1-C10, R is any atom and H is areactive group], or aryl, alkyl and arylalkyl groups that may or may notcrosslink to Cys10 (R306). In some embodiments, R302 is2-carboxy-3-bromo-pyridine, 2-bromo-benzoic acid, 2-bromo-cyclohexanoicacid. R302 is preferably C cross linked to R306, blocked C such as Cmob,cross linked to R306 by a Carba link, any non-crosslinking aa ormimetic.

In some preferred embodiments, R303 is a Cysteine side chain analog[—(CR2)n-S, or —(CR2)n-H; where n is C1-C10, R is any atom and H is areactive group], an alpha, beta or gamma amino acid capable of forming acrosslink with Cys14 (R308) via a side chain functional group (see sidechains, above), an amino acid mimetic, such as2-carboxy-3-thiopiperidine, which constrain the dihedral angles or theside chain to favorable configurations. R303 is preferably C crosslinkedto C at R308 or any other moiety crosslinked to R308.

In some preferred embodiments, R304 is a single amino acid (all types),dipeptide, dipeptide mimetic and chain links, particularly structureswith 4-8 atoms bridging the gap between R303 and R305, most preferablystructures with 6 atoms bridging the gap between R303 and R305.Preferred amino acids for R304 are sarcosine, ornithine and lysine. Glu7preferred amino acid replacements are Gln, Gly, Ser, Ala, Pro, Asp, Asnand L-octahydroindole-2-carboxyilic acid, and their N-alkyl, N-hydroxyl,or N-aryl analogs. Leu8 preferred amino acid replacements are Ile,Norleucine, Met, Val, Phe, Trp, Tyr, Gin, Gly, Ala, Pro andL-1,2,3,4-tetrahydroisoquinoline-3-carboxyilic acid, and their N-alkyl,N-hydroxyl, or N-aryl analogs. R304 is preferably a dipeptide such asE-L, or E-NmethylLeu.

In some preferred embodiments, R305 is a single amino acid (all types),preferably hydrophobic or neutral ones, and their N-alkyl, N-hydroxyl,or N-aryl analogs, more preferably Ala, N(Me)-Ala, Thr, Leu, N(Me)-Leu,Phe and blocked Cysteine, Serine and Threonine, an amino acid mimetic,such as L-1,2,3,4-tetrahydroisoquinoline-3-carboxyilic acid, whichconstrain the dihedral angles or the side chain to favorableconfigurations. In some preferred embodiments, R305 may be comprised ofamino acids or organic molecules with a hydrophobicity of at least −2.0(Glutamic acid is the lowest native AA with a value of −1.22), andpreferably a value greater than zero; and more preferably a valuegreater than one. Hydrophobicity is determined by the method of Sweetand Eisenberg (Sweet R. M., Eisenberg D., J. Mol. Biol.171:479-488(1983) which is incorporated herein by reference). R305 ispreferably A L, T, F, V, I, Y, NmethylLeu, NmethylAlam, and NmethylVal.

In some preferred embodiments, R306 is a single amino acid (all types),or an N-alkyl, N-hydroxyl, or N-aryl analog of one, an amino acidmimetic, such as which constrain the dihedral angles or the side chainto favorable configurations. R306 is preferably C cross linked to R302,blocked C such as Cmob, cross linked to R302 by a Carba link anynon-crosslinking amino acid or mimetic.

In some preferred embodiments, R307 is a dipeptide mimetic, such asbicycle-L-seryl-proline, Btd, APM, ACTB, or ACDN

In some embodiments, R307 is X-Y-Z wherein

X is a single amino acid (all types), preferably Asn, Asp, Ala, His,GlnGly, beta-Alanine), or a short one (Ala, Ser, Thr, Asp), and theirN-alkyl, N-hydroxyl, or N-aryl analogs or a synthetic amino acids,preferably Isoasparagine or beta alanine.

Y is a single amino acids (all types), preferably Pro, Ile, Ala, Val(both D and L) and their N-alkyl, N-hydroxyl, or N-aryl analogs.Synthetic amino acids, such as homopro, Nipecotic acid, isonipecoticacid, Oic, Tic, Aib, aminobenzoate, carboxypiperidine, azetidinecarboxylate and aminocyclopentene carboxylic acid. Amino acid mimetics,such as L-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, whichconstrain the dihedral angles or the side chain to favorableconfigurations.

Z is a single amino acid (all types), preferably with no sidechain (Gly,beta-Alanine), or a short one (Ala, Ser, Thr, Asp), and their N-alkyl,N-hydroxyl, or N-aryl analogs, more preferably Ala, N(Me)-Ala, and Gly.

R307 is preferably a tripeptide N-P-A.

In some preferred embodiments, R308 is a Cysteine side chain analog[—(CR2)n-S, or —(CR2)n-H; where n is C1-C10, R is any atom and H is areactive group], an alpha, beta or gamma amino acids capable of forminga crosslink with Cys6 (R303) via a side chain functional group (see sidechains, above), an amino acid mimetic, such as2-carboxy-3-thiopiperidine, which constrain the dihedral angles or theside chain to favorable configurations. R308 is preferably C crosslinkedto C at R303 or any other moiety crosslinked to R303.

In some preferred embodiments, R309 is preferably smaller than 1000daltons. R9 is preferably 0-20 amino acids, NPAAAGCacmY, or ST11-18 witha change at 17 to remove crosslinking function, COOH, AGA or Y.

In some embodiments, the Loop B compounds either have no tail at R309 orhave one that can be cleaved such that the molecule becomes an agonist.The present invention further relates to Loop B agonist compounds havingFormula (IV):R401-R402-R403-R404-R405-R406-R407-R408-R409  (IV)wherein:

-   -   R401 is combination of 1-50 natural amino acids, 1-50 blocked        amino acids, 1-50 synthetic amino acids, 1-50 derivatized amino        acids; 1-50 amino acid mimetics, a 1-50 alkyls, one or more 1-50        substituted alkyls, one or more 1-50 aryls, one or more 1-50        substituted aryls, one or more 1-50 alkylaryls, one or more 1-50        substituted alkylaryls or combinations thereof;    -   R402 is a natural amino acid, blocked amino acid, synthetic        amino acid, derivatized amino acid, or an amino acid mimetic        wherein R402 may crosslink with R406;    -   R403 is a natural amino acid, blocked amino acid, synthetic        amino acid, derivatized amino acid or amino acid mimetic that        crosslinks with R408;    -   R404 is one or two natural amino acids, one or two blocked amino        acids, one or two synthetic amino acids, one or two derivatized        amino acids, one or two amino acid mimetics, or a dipeptide        mimetic or combinations thereof, wherein R404 forms a 3₁₀-helix        turn group linking R403 to R405;    -   R405 is a natural amino acid, blocked amino acid, synthetic        amino acid, derivatized amino acid or amino acid mimetic that        does not crosslink with R409    -   R406 is a natural amino acid, blocked amino acid, synthetic        amino acid, derivatized amino acid, or amino acid mimetic        wherein R406 may crosslink with R402;    -   R407 is one, two or three natural amino acids, one, two or three        blocked amino acids, one, two or three amino acid mimetics, or a        tripeptide mimetic or combinations thereof, wherein R407 forms a        beta turn group linking R406 to R408    -   R408 is a natural amino acid, blocked amino acid, synthetic        amino acid, derivatized amino acid or amino acid mimetic that        crosslinks with R403;    -   R409 is absent or 1-50 natural amino acids, 1-50 blocked amino        acids, 1-50 synthetic amino acids, 1-50 derivatized amino acids;        1-50 amino acid mimetics, a 1-50 alkyls, one or more 1-50        substituted alkyls, one or more 1-50 aryls, one or more 1-50        substituted aryls, one or more 1-50 alkylaryls, one or more 1-50        substituted alkylaryls or combinations thereof that does not        crosslink to R405.

In some preferred embodiments, R401 is preferably 1-50 natural aminoacids, 1-50 blocked amino acids, 1-50 synthetic amino acids, 1-50derivatized amino acids; 1-50 amino acid mimetics, a 1-50 alkyls, one ormore 1-50 substituted alkyls, one or more 1-50 aryls, one or more 1-50substituted aryls, one or more 1-50 alkylaryls, one or more 1-50substituted alkylaryls or combinations thereof. In some embodiments,R401 is 0-20 amino acids. In some embodiments R401 is smaller than 1000daltons. In some embodiments, R401 is Y, NTFY, SSSNY, SSDWDY, ID, PN, orQE.

In some preferred embodiments, R402 is a Cysteine side chain analog[—(CR2)n-S, or —(CR2)n-H; where n is C1-C10, R is any atom and H is areactive group], or aryl, alkyl and arylalkyl groups that may or may notcrosslink to Cys10 (R406). In some embodiments, R402 is2-carboxy-3-bromo-pyridine, 2-bromo-benzoic acid, 2-bromo-cyclohexanoicacid. R402 is preferably C cross linked to R406, blocked C such as Cmob,cross linked to R406 by a Carba link, any non-crosslinking aa ormimetic.

In some preferred embodiments, R403 is a Cysteine side chain analog[—(CR2)n-S, or —(CR2)n-H; where n is C1-C10, R is any atom and H is areactive group], an alpha, beta or gamma amino acid capable of forming acrosslink with Cys14 (R408) via a side chain functional group (see sidechains, above), an amino acid mimetic, such as2-carboxy-3-thiopiperidine, which constrain the dihedral angles or theside chain to favorable configurations. R403 is preferably C crosslinkedto C at R408 or any other moiety crosslinked to R408.

In some preferred embodiments, R404 is a single amino acid (all types),dipeptide, dipeptide mimetic and chain links, particularly structureswith 4-8 atoms bridging the gap between R403 and R405, most preferablystructures with 6 atoms bridging the gap between R403 and R405.Preferred amino acids for R404 are sarcosine, ornithine and lysine. Glu7preferred amino acid replacements are Gln, Gly, Ser, Ala, Pro, Asp, Asnand L-octahydroindole-2-carboxyilic acid, and their N-alkyl, N-hydroxyl,or N-aryl analogs. Leu8 preferred amino acid replacements are Ile,Norleucine, Met, Val, Phe, Trp, Tyr, Gln, Gly, Ala, Pro andL-1,2,3,4-tetrahydroisoquinoline-3-carboxyilic acid, and their N-alkyl,N-hydroxyl, or N-aryl analogs. R404 is preferably a dipeptide such asE-L, or E-NmethylLeu.

In some preferred embodiments, R405 is a single amino acid (all types),preferably hydrophobic or neutral ones, and their N-alkyl, N-hydroxyl,or N-aryl analogs, more preferably Ala, N(Me)-Ala, Thr, Leu, N(Me)-Leu,Phe and blocked Cysteine, Serine and Threonine, an amino acid mimetic,such as L-1,2,3,4-tetrahydroisoquinoline-3-carboxyilic acid, whichconstrain the dihedral angles or the side chain to favorableconfigurations. In some preferred embodiments, R405 may be comprised ofamino acids or organic molecules with a hydrophobicity of at least −2.0(Glutamic acid is the lowest native AA with a value of −1.22), andpreferably a value greater than zero; and more preferably a valuegreater than one. Hydrophobicity is determined by the method of Sweetand Eisenberg (Sweet R. M., Eisenberg D., J. Mol. Biol. 171:479488(1983)which is incorporated herein by reference). R405 is preferably A L, T,F, V, I, Y, NmethylLeu, NmethylAlam, and NmethylVal.

In some preferred embodiments, R406 is a single amino acid (all types),or an N-alkyl, N-hydroxyl, or N-aryl analog of one, an amino acidmimetic, such as which constrain the dihedral angles or the side chainto favorable configurations. R406 is preferably C cross linked to R402,blocked C such as Cmob, cross linked to R402 by a Carba link anynon-crosslinking amino acid or mimetic.

In some preferred embodiments, R407 is a dipeptide mimetic, such asbicycle-L-seryl-proline, Btd, APM, ACTB, or ACDN

In some embodiments, R407 is X-Y-Z wherein

X is a single amino acid (all types), preferably Asn, Asp, Ala, His,GlnGly, beta-Alanine), or a short one (Ala, Ser, Thr, Asp), and theirN-alkyl, N-hydroxyl, or N-aryl analogs or a synthetic amino acids,preferably Isoasparagine or beta alanine.

Y is a single amino acids (all types), preferably Pro, Ile, Ala, Val(both D and L) and their N-alkyl, N-hydroxyl, or N-aryl analogs.Synthetic amino acids, such as homopro, Nipecotic acid, isonipecoticacid, Oic, Tic, Aib, aminobenzoate, carboxypiperidine, azetidinecarboxylate and aminocyclopentene carboxylic acid. Amino acid mimetics,such as L-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, whichconstrain the dihedral angles or the side chain to favorableconfigurations.

Z is a single amino acid (all types), preferably with no sidechain (Gly,beta-Alanine), or a short one (Ala, Ser, Thr, Asp), and their N-alkyl,N-hydroxyl, or N-aryl analogs, more preferably Ala, N(Me)-Ala, and Gly.

R407 is preferably a tripeptide N-P-A.

In some preferred embodiments, R408 is a Cysteine side chain analog[—(CR2)n-S, or (CR2)n-H; where n is C1-C10, R is any atom and H is areactive group], an alpha, beta or gamma amino acids capable of forminga crosslink with Cys6 (R403) via a side chain functional group (see sidechains, above), an amino acid mimetic, such as2-carboxy-3-thiopiperidine, which constrain the dihedral angles or theside chain to favorable configurations. R408 is preferably C crosslinkedto C at R403 or any other moiety crosslinked to R403.

In some preferred embodiments, R409 is preferably COOH. In someembodiments, R9 is preferably 0-20 amino acids, provided that it iscleavable in vivo to be converted to an agonist. Examples of cleavabletails are NPAAAGCacmY, and ST11-18 with a change at 17 to removecrosslinking function.

The present invention also relates to compounds that are Loop Bprodrugs, that is Loop B compounds which have tails that render themcompound of the inventions which tails are claeavable. The presentinvention relates to compounds having Formula (V): Formula (V):R501-R502-R503-R504-R505-R506-R507-R508-R509  (V)wherein:

-   -   R501 is combination of 1-50 natural amino acids, 1-50 blocked        amino acids, 1-50 synthetic amino acids, 1-50 derivatized amino        acids; 1-50 amino acid mimetics, a 1-50 alkyls, one or more 1-50        substituted alkyls, one or more 1-50 aryls, one or more 1-50        substituted aryls, one or more 1-50 alkylaryls, one or more 1-50        substituted alkylaryls or combinations thereof;    -   R502 is a natural amino acid, blocked amino acid, synthetic        amino acid, derivatized amino acid, or an amino acid mimetic        wherein R502 may crosslink with R506;    -   R503 is a natural amino acid, blocked amino acid, synthetic        amino acid, derivatized amino acid or amino acid mimetic that        crosslinks with R508;    -   R504 is one or two natural amino acids, one or two blocked amino        acids, one or two synthetic amino acids, one or two derivatized        amino acids, one or two amino acid mimetics, or a dipeptide        mimetic or combinations thereof, wherein R504 forms a 3₁₀-helix        turn group linking R503 to R505;    -   R505 is a natural amino acid, blocked amino acid, synthetic        amino acid, derivatized amino acid or amino acid mimetic that        does not crosslink with R509    -   R506 is a natural amino acid, blocked amino acid, synthetic        amino acid, derivatized amino acid, or amino acid mimetic        wherein R506 may crosslink with R502;    -   R507 is one, two or three natural amino acids, one, two or three        blocked amino acids, one, two or three amino acid mimetics, or a        tripeptide mimetic or combinations thereof, wherein R507 forms a        beta turn group linking R506 to R508    -   R508 is a natural amino acid, blocked amino acid, synthetic        amino acid, derivatized amino acid or amino acid mimetic that        crosslinks with R503;    -   R509 is 1-50 natural amino acids, 1-50 blocked amino acids, 1-50        synthetic amino acids, 1-50 derivatized amino acids; 1-50 amino        acid mimetics, a 1-50 alkyls, one or more 1-50 substituted        alkyls, one or more 1-50 aryls, one or more 1-50 substituted        aryls, one or more 1-50 alkylaryls, one or more 1-50 substituted        alkylaryls or combinations thereof that does not crosslink to        R505;    -   wherein the compound is degradable in vivo and the structure of        the compounds is not C-C-E-L-A-C-N-P-A-C-T-G-A.

In some preferred embodiments, R501 is preferably 1-50 natural aminoacids, 1-50 blocked amino acids, 1-50 synthetic amino acids, 1-50derivatized amino acids; 1-50 amino acid mimetics, a 1-50 alkyls, one ormore 1-50 substituted alkyls, one or more 1-50 aryls, one or more 1-50substituted aryls, one or more 1-50 alkylaryls, one or more 1-50substituted alkylaryls or combinations thereof. In some embodiments,R501 is 0-20 amino acids. In some embodiments R501 is smaller than 1000daltons. In some embodiments, R501 is Y, NTFY, SSSNY, SSDWDY, ID, PN, orQE.

In some preferred embodiments, R502 is a Cysteine side chain analog[—(CR2)n-S, or —(CR2)n-H; where n is C1-C10, R is any atom and H is areactive group], or aryl, alkyl and arylalkyl groups that may or may notcrosslink to Cys10 (R506). In some embodiments, R502 is2-carboxy-3-bromo-pyridine, 2-bromo-benzoic acid, 2-bromo-cyclohexanoicacid. R502 is preferably C cross linked to R506, blocked C such as Cmob,cross linked to R506 by a Carba link, any non-crosslinking aa ormimetic.

In some preferred embodiments, R503 is a Cysteine side chain analog[—(CR2)n-S, or —(CR2)n-H; where n is C1-C10, R is any atom and H is areactive group], an alpha, beta or gamma amino acid capable of forming acrosslink with Cys14 (R508) via a side chain functional group (see sidechains, above), an amino acid mimetic, such as2-carboxy-3-thiopiperidine, which constrain the dihedral angles or theside chain to favorable configurations. R503 is preferably C crosslinkedto C at R508 or any other moiety crosslinked to R508.

In some preferred embodiments, R504 is a single amino acid (all types),dipeptide, dipeptide mimetic and chain links, particularly structureswith 4-8 atoms bridging the gap between R503 and R505, most preferablystructures with 6 atoms bridging the gap between R503 and R505.Preferred amino acids for R504 are sarcosine, ornithine and lysine. Glu7preferred amino acid replacements are Gln, Gly, Ser, Ala, Pro, Asp, Asnand L-octahydroindole-2-carboxyilic acid, and their N-alkyl, N-hydroxyl,or N-aryl analogs. Leu8 preferred amino acid replacements are Ile,Norleucine, Met, Val, Phe, Trp, Tyr, Gln, Gly, Ala, Pro andL-1,2,3,4-tetrahydroisoquinoline-3-carboxyilic acid, and their N-alkyl,N-hydroxyl, or N-aryl analogs. R504 is preferably a dipeptide such asE-L, or E-NmethylLeu.

In some preferred embodiments, R505 is a single amino acid (all types),preferably hydrophobic or neutral ones, and their N-alkyl, N-hydroxyl,or N-aryl analogs, more preferably Ala, N(Me)-Ala, Thr, Leu, N(Me)-Leu,Phe and blocked Cysteine, Serine and Threonine, an amino acid mimetic,such as L-1,2,3,4-tetrahydroisoquinoline-3-carboxyilic acid, whichconstrain the dihedral angles or the side chain to favorableconfigurations. In some preferred embodiments, R505 may be comprised ofamino acids or organic molecules with a hydrophobicity of at least −2.0(Glutamic acid is the lowest native AA with a value of −1.22), andpreferably a value greater than zero; and more preferably a valuegreater than one. Hydrophobicity is determined by the method of Sweetand Eisenberg (Sweet R. M., Eisenberg D., J. Mol. Biol.171:479-488(1983) which is incorporated herein by reference). R505 ispreferably A L, T, F, V, I, Y, NmethylLeu, NmethylAlam, and NmethylVal.

In some preferred embodiments, R506 is a single amino acid (all types),or an N-alkyl, N-hydroxyl, or N-aryl analog of one, an amino acidmimetic, such as which constrain the dihedral angles or the side chainto favorable configurations. R506 is preferably C cross linked to R502,blocked C such as Cmob, cross linked to R502 by a Carba link anynon-crosslinking amino acid or mimetic.

In some preferred embodiments, R507 is a dipeptide mimetic, such asbicycle-L-seryl-proline, Btd, APM, ACTB, or ACDN

In some embodiments, R507 is X-Y-Z wherein

X is a single amino acid (all types), preferably Asn, Asp, Ala, His,GlnGly, beta-Alanine), or a short one (Ala, Ser, Thr, Asp), and theirN-alkyl, N-hydroxyl, or N-aryl analogs or a synthetic amino acids,preferably Isoasparagine or beta alanine.

Y is a single amino acids (all types), preferably Pro, Ile, Ala, Val(both D and L) and their N-alkyl, N-hydroxyl, or N-aryl analogs.Synthetic amino acids, such as homopro, Nipecotic acid, isonipecoticacid, Oic, Tic, Aib, aminobenzoate, carboxypiperidine, azetidinecarboxylate and aminocyclopentene carboxylic acid. Amino acid mimetics,such as L-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, whichconstrain the dihedral angles or the side chain to favorableconfigurations.

Z is a single amino acid (all types), preferably with no sidechain (Gly,beta-Alanine), or a short one (Ala, Ser, Thr, Asp), and their N-alkyl,N-hydroxyl, or N-aryl analogs, more preferably Ala, N(Me)-Ala, and Gly.

R507 is preferably a tripeptide N-P-A.

In some preferred embodiments, R508 is a Cysteine side chain analog[—(CR2)n-S, or —(CR2)n-H; where n is C1-C10, R is any atom and H is areactive group], an alpha, beta or gamma amino acids capable of forminga crosslink with Cys6 (R503) via a side chain functional group (see sidechains, above), an amino acid mimetic, such as2-carboxy-3-thiopiperidine, which constrain the dihedral angles or theside chain to favorable configurations. R508 is preferably C crosslinkedto C at R503 or any other moiety crosslinked to R503.

In some preferred embodiments, R509 is preferably cleavable or otherwisedegradable in vivo such that it is converted from a compound of theinvention to an agonist. Examples of cleavable tails are NPAAAGCacmY,and ST 11-18 with a change at 17 to remove crosslinking function.

The present invention also relates to compounds that are Loop B compoundof the inventions which are Loop B compounds that have nondegardabletails. The present invention relates to compounds having Formula (VI):R601-R602-R603-R604-R605-R606-R607-R608-R609  (VI)wherein:

-   -   R601 is combination of 1-50 natural amino acids, 1-50 blocked        amino acids, 1-50 synthetic amino acids, 1-50 derivatized amino        acids; 1-50 amino acid mimetics, a 1-50 alkyls, one or more 1-50        substituted alkyls, one or more 1-50 aryls, one or more 1-50        substituted aryls, one or more 1-50 alkylaryls, one or more 1-50        substituted alkylaryls or combinations thereof;    -   R602 is a natural amino acid, blocked amino acid, synthetic        amino acid, derivatized amino acid, or an amino acid mimetic        wherein R602 may crosslink with R606;    -   R603 is a natural amino acid, blocked amino acid, synthetic        amino acid, derivatized amino acid or amino acid mimetic that        crosslinks with R608;    -   R604 is one or two natural amino acids, one or two blocked amino        acids, one or two synthetic amino acids, one or two derivatized        amino acids, one or two amino acid mimetics, or a dipeptide        mimetic or combinations thereof, wherein R604 forms a 3₁₀-helix        turn group linking R603 to R605;    -   R605 is a natural amino acid, blocked amino acid, synthetic        amino acid, derivatized amino acid or amino acid mimetic that        does not crosslink with R609    -   R606 is a natural amino acid, blocked amino acid, synthetic        amino acid, derivatized amino acid, or amino acid mimetic        wherein R606 may crosslink with R602;    -   R607 is one, two or three natural amino acids, one, two or three        blocked amino acids, one, two or three amino acid mimetics, or a        tripeptide mimetic or combinations thereof, wherein R607 forms a        beta turn group linking R606 to R608    -   R608 is a natural amino acid, blocked amino acid, synthetic        amino acid, derivatized amino acid or amino acid mimetic that        crosslinks with R603;    -   R609 is 1-50 natural amino acids, 1-50 blocked amino acids, 1-50        synthetic amino acids, 1-50 derivatized amino acids; 1-50 amino        acid mimetics, a 1-50 alkyls, one or more 1-50 substituted        alkyls, one or more 1-50 aryls, one or more 1-50 substituted        aryls, one or more 1-50 alkylaryls, one or more 1-50 substituted        alkylaryls or combinations thereof that does not crosslink to        R605 and is non-degradable in vivo.

In some preferred embodiments, R601 is preferably 1-50 natural aminoacids, 1-50 blocked amino acids, 1-50 synthetic amino acids, 1-50derivatized amino acids; 1-50 amino acid mimetics, a 1-50 alkyls, one ormore 1-50 substituted alkyls, one or more 1-50 aryls, one or more 1-50substituted aryls, one or more 1-50 alkylaryls, one or more 1-50substituted alkylaryls or combinations thereof. In some embodiments,R601 is 0-20 amino acids. In some embodiments R601 is smaller than 1000daltons. In some embodiments, R601 is Y, NTFY, SSSNY, SSDWDY, ID, PN, orQE.

In some preferred embodiments, R602 is a Cysteine side chain analog[—(CR2)n-S, or —(CR2)n-H; where n is C1-C10, R is any atom and H is areactive group], or aryl, alkyl and arylalkyl groups that may or may notcrosslink to Cys10 (R606). In some embodiments, R602 is2-carboxy-3-bromo-pyridine, 2-bromo-benzoic acid, 2-bromo-cyclohexanoicacid. R602 is preferably C cross linked to R606, blocked C such as Cmob,cross linked to R606 by a Carba link, any non-crosslinking aa ormimetic.

In some preferred embodiments, R603 is a Cysteine side chain analog[—(CR2)n-S, or —(CR2)n-H; where n is C1-C10, R is any atom and H is areactive group], an alpha, beta or gamma amino acid capable of forming acrosslink with Cys14 (R608) via a side chain functional group (see sidechains, above), an amino acid mimetic, such as2-carboxy-3-thiopiperidine, which constrain the dihedral angles or theside chain to favorable configurations. R603 is preferably C crosslinkedto C at R608 or any other moiety crosslinked to R608.

In some preferred embodiments, R604 is a single amino acid (all types),dipeptide, dipeptide mimetic and chain links, particularly structureswith 4-8 atoms bridging the gap between R603 and R605, most preferablystructures with 6 atoms bridging the gap between R603 and R605.Preferred amino acids for R604 are sarcosine, ornithine and lysine. Glu7preferred amino acid replacements are Gln, Gly, Ser, Ala, Pro, Asp, Asnand L-octahydroindole-2-carboxyilic acid, and their N-alkyl, N-hydroxyl,or N-aryl analogs. Leu8 preferred amino acid replacements are Ile,Norleucine, Met, Val, Phe, Trp, Tyr, Gln, Gly, Ala, Pro andL-1,2,3,4-tetrahydroisoquinoline-3-carboxyilic acid, and their N-alkyl,N-hydroxyl, or N-aryl analogs. R604 is preferably a dipeptide such asE-L, or E-NmethylLeu.

In some preferred embodiments, R605 is a single amino acid (all types),preferably hydrophobic or neutral ones, and their N-alkyl, N-hydroxyl,or N-aryl analogs, more preferably Ala, N(Me)-Ala, Thr, Leu, N(Me)-Leu,Phe and blocked Cysteine, Serine and Threonine, an amino acid mimetic,such as L-1,2,3,4-tetrahydroisoquinoline-3-carboxyilic acid, whichconstrain the dihedral angles or the side chain to favorableconfigurations. In some preferred embodiments, R605 may be comprised ofamino acids or organic molecules with a hydrophobicity of at least −2.0(Glutamic acid is the lowest native AA with a value of −1.22), andpreferably a value greater than zero; and more preferably a valuegreater than one. Hydrophobicity is determined by the method of Sweetand Eisenberg (Sweet R. M., Eisenberg D., J. Mol. Biol.171:479-488(1983) which is incorporated herein by reference). R605 ispreferably A L, T, F, V, I, Y, NmethylLeu, NmethylAlam, and NmethylVal.

In some preferred embodiments, R606 is a single amino acid (all types),or an N-alkyl, N-hydroxyl, or N-aryl analog of one, an amino acidmimetic, such as which constrain the dihedral angles or the side chainto favorable configurations. R606 is preferably C cross linked to R602,blocked C such as Cmob, cross linked to R602 by a Carba link anynon-crosslinking amino acid or mimetic.

In some preferred embodiments, R607 is a dipeptide mimetic, such asbicycle-L-seryl-proline, Btd, APM, ACTB, or ACDN

In some embodiments, R607 is X-Y-Z wherein

X is a single amino acid (all types), preferably Asn, Asp, Ala, His,GlnGly, beta-Alanine), or a short one (Ala, Ser, Thr, Asp), and theirN-alkyl, N-hydroxyl, or N-aryl analogs or a synthetic amino acids,preferably Isoasparagine or beta alanine.

Y is a single amino acids (all types), preferably Pro, Ile, Ala, Val(both D and L) and their N-alkyl, N-hydroxyl, or N-aryl analogs.Synthetic amino acids, such as homoPro, Nipecotic acid, isonipecoticacid, Oic, Tic, Aib, aminobenzoate, carboxypiperidine, azetidinecarboxylate and aminocyclopentene carboxylic acid. Amino acid mimetics,such as L-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, whichconstrain the dihedral angles or the side chain to favorableconfigurations.

Z is a single amino acid (all types), preferably with no sidechain (Gly,beta-Alanine), or a short one (Ala, Ser, Thr, Asp), and their N-alkyl,N-hydroxyl, or N-aryl analogs, more preferably Ala, N(Me)-Ala, and Gly.

R607 is preferably a tripeptide N-P-A.

In some preferred embodiments, R608 is a Cysteine side chain analog[—(CR2)n-S, or —(CR2)n-H; where n is C1-C10, R is any atom and H is areactive group], an alpha, beta or gamma amino acids capable of forminga crosslink with Cys6 (R503) via a side chain functional group (see sidechains, above), an amino acid mimetic, such as2-carboxy-3-thiopiperidine, which constrain the dihedral angles or theside chain to favorable configurations. R608 is preferably C crosslinkedto C at R603 or any other moiety crosslinked to R603.

In some preferred embodiments, R609 is non-degradable in vivo such thatit is not converted from a compound of the invention to an agonist.Examples of cleavable tails are organics or non-naturally occurringamino-acids.

Compounds may be produced synthetically or recombinantly. Peptides andconjugated compositions or portions thereof which are peptides may beprepared using the solid-phase synthetic technique initially describedby Merrifield, in J. Am. Chem. Soc., 15:2149-2154 (1963). Other peptidesynthesis techniques may be found, for example, in M. Bodanszky et al.,(1976) Peptide Synthesis, John Wiley & Sons, 2d Ed.; Kent andClark-Lewis in Synthetic Peptides in Biology and Medicine, p. 295-358,eds. Alitalo, K., et al. Science Publishers, (Amsterdam, 1985); as wellas other reference works known to those skilled in the art. A summary ofpeptide synthesis techniques may be found in J. Stuart and J. D. Young,Solid Phase Peptide Synthelia, Pierce Chemical Company, Rockford, Ill.(1984), which is incorporated herein by reference. The synthesis ofpeptides by solution methods may also be used, as described in TheProteins, Vol. II, 3d Ed., p. 105-237, Neurath, H. et al., Eds.,Academic Press, New York, N.Y. (1976). Appropriate protective groups foruse in such syntheses will be found in the above texts, as well as in J.F. W. McOmie, Protective Groups in Organic Chemistry, Plenum Press, NewYork, N.Y. (1973), which is incorporated herein by reference. Ingeneral, these synthetic methods involve the sequential addition of oneor more amino acid residues or suitable protected amino acid residues toa growing peptide chain. Normally, either the amino or carboxyl group ofthe first amino acid residue is protected by a suitable, selectivelyremovable protecting group. A different, selectively removableprotecting group is utilized for amino acids containing a reactive sidegroup, such as lysine.

Using a solid phase synthesis as an example, the protected orderivatized amino acid is attached to an inert solid support through itsunprotected carboxyl or amino group. The protecting group of the aminoor carboxyl group is then selectively removed and the next amino acid inthe sequence having the complementary (amino or carboxyl) group suitablyprotected is admixed and reacted with the residue already attached tothe solid support. The protecting group of the amino or carboxyl groupis then removed from this newly added amino acid residue, and the nextamino acid (suitably protected) is then added, and so forth. After allthe desired amino acids have been linked in the proper sequence, anyremaining terminal and side group protecting groups (and solid support)are removed sequentially or concurrently, to provide the final peptide.The peptide of the invention are preferably devoid of benzylated ormethylbenzylated amino acids. Such protecting group moieties may be usedin the course of synthesis, but they are removed before the peptides areused. Additional reactions may be necessary, as described elsewhere, toform intramolecular linkages to restrain conformation.

According to some embodiments of the invention, compounds of theinvention are conjugated to a detectable moiety, making them particularuseful as imaging agents or diagnostic test reagents. Detectablemoieties include radioactive as well as non-radioactive moieties.

Examples of radioactive include substitution of atoms of the compoundwith a radioactive isotope of such atoms. Alternatively, the radioactivemoiety may be a radionuclide or a radioactive compound which isconjugated to the compound, either directly or through use of a linker.Examples of non-radioactive compounds include a GCC compound of theinvention conjugated to a detectable, non-radioactive moiety. In someembodiments the GCC compound of the invention is linked to a linker thatforms a bond or complex with a detectable moiety. include anon-radioactive compound which is conjugated to the compound, eitherdirectly or through use of a linker.

Examples of moieties which can be conjugated to the compounds of theinvention of include a radionuclide, such as for example, ¹²⁵I, ⁴³K,⁵²Fe, ⁵⁷Co, ⁶⁷CU, ⁶⁷Ga, ⁶⁸Ga, ⁷⁷Br, ⁸¹ Rb/⁸¹MK, ⁸⁷MSr, ⁹⁹MTc, ¹¹¹n,¹¹³MIn, ¹²³I, ¹²⁵I, ¹²⁷Cs, ¹²⁹Cs, ¹³¹I, ¹³²I, ¹⁹⁷Hg, ²⁰³Pb and ²⁰⁶206Bi,⁴⁷Sc, ⁶⁷Cu, ⁹⁰Y, ¹⁰⁹Pd, ¹²³I, ¹²⁵I, ¹³¹I, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁹⁹AU, ²¹¹At,²¹²Pb and ²¹²Bi, ³²P and ³³P, ⁷¹Ge ⁷⁷As, ¹⁰³Pb, ¹¹¹Ag, ¹¹⁹Sb, ¹²¹Sn,¹³¹Cs, ¹⁴³Pr, ¹⁶¹Tb, ¹⁷⁷Lu, ¹⁹¹Os, ¹⁹³Pt, ¹⁹⁷Hg; beta negative and/orauger emitters; enzymes, such as for example, malate dehydrogenase,staphylococcal nuclease, delta-5-steroid isomerase, yeast alcoholdehydrogenase, alpha-glycerophosphate dehydrogenase, triose phosphateisomerase, horseradish peroxidase, alkaline phosphatase, asparaginase,glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase,glucose-6-phosphate dehydrogenase, glucoamylase andacetylcholinesterase, fluorescent labels, such as for example,fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin,allophycocyanin, o-phthaldehyde and fluorescamine; fluorescence-emittingmetals such as ¹⁵²Eu, or others of the lanthanide series; metals usingsuch metal chelating groups as diethylenetriaminepentaacetic acid (DTPA)or ethylenediamine-tetraacetic acid (EDTA); chemiluminescent labels suchas for example luminol, isoluminol, theromatic acridinium ester,imidazole, acridinium salt and oxalate ester; and bioluminescent labelssuch as for example luciferin, luciferase and aequorin. Examples alsoinclude chemotherapeutics, such as for example, methotrexate(amethopterin), doxorubicin (adrimycin), daunorubicin,cytosinarabinoside, etoposide, 5-4 fluorouracil, melphalan,chlorambucil, and other nitrogen mustards (e.g. cyclophosphamide),cis-platinum, vindesine (and other vinca alkaloids), mitomycin andbleomycin. Other chemotherapeutics include: purothionin (barley flouroligopeptide), macromomycin. 1,4-benzoquinone derivatives and trenimon;toxins such as for examples ricin, ricin A chain (ricin toxin),Pseudomonas exotoxin (PE), diphtheria toxin (DT), Clostridiumperfringens phospholipase C (PLC), bovine pancreatic ribonuclease (BPR),pokeweed antiviral protein (PAP), abrin, abrin A chain (abrin toxin),cobra venom factor (CVF), gelonin (GEL), saporin (SAP), modeccin,viscumin and volkensin; inactive prodrug which can be converted by theenzyme into an active drug such as a prodrug, such as for exampleetoposidephosphate, that can be converted to an active drug by an enzymesuch as for example alkaline phosphatase/etoposidephosphate;radiosensitizing agents such as for example nitroimidazoles,metronidazole and misonidazole; radionuclides useful as toxins inradiation therapy such as for example ⁴⁷Sc, ⁶⁷CU, ⁹⁰Y, ¹⁰⁹Pd, ¹²³I,¹²⁵I, ¹³¹I, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁹⁹Au, ²¹¹At, ²¹²Pb and ²¹²Bi, ³²P and ³³P,⁷¹Ge, ⁷⁷As, ¹⁰³Pb, 105 Rh, ¹¹¹Ag, ¹¹⁹Sb, ¹²¹Sn, ¹³¹Cs, ¹⁴³Pr, ¹⁶¹Tb,¹⁷⁷Lu, ¹⁹¹Os, ¹⁹³ MPt, ¹⁹⁷Hg; and all beta negative and/or augeremitters; heavy metal such as iron chelates, chelates of gadolinium ormanganese; positron emitters of oxygen, nitrogen, iron, carbon, orgallium; radionuclides useful in imaging procedures such as for example⁴³K, ⁵²Fe, ⁵⁷Co, ⁶⁷Cu, ⁶⁷Ga, ⁶⁸Ga, ⁷⁷Br, ⁸¹Rb/⁸¹MKr, ⁸⁷MSr, ⁹⁹MTc,¹¹¹In, ¹¹³MIn, ¹²³I, ¹²⁵I, ²⁷Cs, ¹²⁹Cs, ¹³¹I, ¹³²I, ⁹⁷Hg, ²⁰³Pb and²⁰⁶206Bi. Additional examples include photodynamic agents such asfluorophore or porphyrins. Porphyrin may include hematoporphyrinderivative (HPD) and porfimer sodium (Photofrin.RTM.). A secondgeneration photosensitizers is BPD verteporfin. In some embodiments thefluorophore is tetramethylrotamine.

Compositions of the Invention

According to some embodiments of the invention, compositions areprovided which comprise conjugated or non-conjugated compounds as wellas kits.

Compositions comprising conjugated compounds useful for in vivo imagingor therapeutics are provided as pharmaceutical compositions. In someembodiments, the compounds are provided as pharmaceuticals adapted fororal administration. In such embodiments, the composition may be in theform of a tablet, capsule or liquid. In some embodiments, the tablet orcapsule may be enterically coated or otherwise adapted to pass thestomach intact for delivery to the intestine. In some embodiments, thecompositions are sterile, pyrogen free compositions particularlysuitable as injectables. Some may be adapted for topical applicationsuch as spray on imaging compositions. In some embodiments, thepharmaceutical compositions comprise the compounds of the invention incombination with a liposome comprising a therapeutic compound. In somepreferred embodiments, the compound of the invention is provided on theexterior of a liposome that comprises a chemotherapeutic, genetherapeutic, antisense compound, toxin or radioactive substance.

In some embodiments, the compound is provided in a composition adaptedfor use as an in vitro/ex vivo reagent such as a diagnostic reagent.

The pharmaceutical composition of the present invention may beformulated by one having ordinary skill in the art. Suitablepharmaceutical carriers are described in Remington's PharmaceuticalSciences, A. Osol, a standard reference text in this field, which isincorporated herein by reference. In carrying out methods of the presentinvention, unconjugated and conjugated compounds of the presentinvention can be used alone or in combination with other diagnostic,therapeutic or additional agents. Such additional agents includeexcipients such as coloring, stabilizing agents, osmotic agents andantibacterial agents.

For parenteral administration, the peptides of the invention can be, forexample, formulated as a solution, suspension, emulsion or lyophilizedpowder in association with a pharmaceutically acceptable parenteralvehicle. Examples of such vehicles are water, saline, Ringer's solution,dextrose solution, and 5% human serum albumin. Liposomes and nonaqueousvehicles such as fixed oils may also be used. The vehicle or lyophilizedpowder may contain additives that maintain isotonicity (e.g., sodiumchloride, mannitol) and chemical stability (e.g., buffers andpreservatives). The formulation is sterilized by commonly usedtechniques. The compositions are made free of pyrogens by commonly usedtechniques. For example, a parenteral composition suitable foradministration by injection is prepared by dissolving 1.5% by weight ofactive ingredient in 0.9% sodium chloride solution.

The pharmaceutical compositions of the present invention may beadministered by any means that enables the active agent to reach thetargeted cells. These methods include, but are not limited to, oral,topical, intradermal, subcutaneous, intravenous, intramuscular andintraparenteral modes of administration. The compounds may beadministered singly or in combination with other compounds. Thecompounds of the invention are preferably administered with apharmaceutically acceptable carrier selected on the basis of theselected route of administration and standard pharmaceutical practice.

Methods of Diagnosing, Imaging and Treating Cancer

The present invention relates to compounds and methods for in vivoimaging and treatment of tumors originating from the alimentary canal,particularly Barrett's esophagus, primary and metastatic stomach andesophageal tumors and metastatic colorectal tumors.

Carcinomas derived from the colorectal cells, stomach or esophagusexpress GCC. The expression of GCC by such tumors enables this proteinto be a specific biomarker for the presence of cancer cells inextra-intestinal tissues and blood. Indeed, this characteristic permitsthe detection of GCC using the compounds of the invention in adiagnostic test to diagnose and stage patients with colorectal, stomachor esophageal cancer and follow patients after surgery for evidence ofrecurrent disease in their blood as well as to detect colorectal,stomach and esophageal cancers. Further, the GCC may be targeted withsome embodiments of the invention in order to deliver the active agent,such as a detectable agent or a chemotherapeutic to tumor cells in vivo.

Detection of the expression of GCC employing molecular techniques can beemployed to diagnose and stage patients, follow the development ofrecurrence after surgery and/or remission, and, potentially, screennormal people for the development of colorectal, stomach or esophagealcancer.

In individuals suffering from colorectal cancer, the cancer cells areoften derived from cells that produce and display the GCC and thesecancer cells continue to produce GCC. It has been observed that GCC isexpressed by colorectal cancer cells. Likewise, GCC is expressed bystomach and esophageal cancer cells.

The expression of GCC by colorectal tumor cells provides a detectabletarget for in vitro screening, monitoring and staging as well as atarget for in vivo delivery of conjugated compositions that compriseactive agents for the imaging and treatment.

The expression of GCC by stomach and esophageal tumor cells provides adetectable target for in vitro screening, monitoring and staging as wellas a target for in vivo delivery of conjugated compositions thatcomprise active agents for the imaging and treatment.

The expression of GCC by esophageal cells is indicative of Barrett'sesophagus. GCC provides a detectable target for in vitro screening,monitoring and staging as well as a target for in vivo delivery ofconjugated compositions that comprise active agents for the imaging andtreatment.

Preferred Uses

Compounds according to the invention that are GCC compound of theinventions bind to GCC but do not activate the GCC signal pathway.Accordingly, such compounds prevent diarrhea in humans that is inducedby ST producing organisms, which is especially relevant in developingcountries where such organisms are common. By administering compoundsprophylactically on a regular basis, such compounds can be used toprevent diarrhea and the growth retardation that occurs in children indeveloping countries due to chronic ST-induced diarrhea. The compoundsare particularly useful to prevent travelers' diarrhea when taken byindividuals who will be exposed to organisms which produce ST such asthose traveling to countries where such organisms are endemic. Thecompounds are useful to prevent scours and other diarrheal diseases inanimals.

The compounds are also useful therapeutically. For example, thecompounds may be used to treat diarrhea known to be ST induced as wellas secretory diarrhea of unknown etiology. Similarly, they may be usedto treat irritable bowel syndrome and other forms of diarrhea including,but not limited to, diarrhea associated with inflammatory bowel disease,sprue, etc.

Because compounds of formula bind to GCC, the may be used to detect,image or treat cancer. Compounds may be used in methods for earlydetection of neoplastic transformation within the upper GI-tract as aligand to identify GCC expression. Similarly, screening, diagnosis,staging and post-operative surveillance of colorectal tumors may beundertaken using compounds which serve as a ligand to identify GCCexpression. The compounds may be used in diagnostic and therapeutictargeting of primary and metastatic esophageal and gastric tumors andmetastatic colorectal tumors as a ligand for GCCm and to facilitateintestinal recovery from insult e.g. intestinal adaptation, wherein theepithelium requires regeneration including, but not limited to, ischemicinsult, chemical insult, chemotherapy, trauma, and surgery.

Compounds according to the invention that are GCC agonists bind to GCCand activate the GCC signal pathway. Accordingly, such compounds treatBarrett's esophagus as well as primary and metastatic stomach andesophageal cancer as well as metastatic cancer. The compounds are usefultherapeutically alone or in combination with other therapeutic agents.

Because compounds of formula bind to GCC, the may be used to detect,image or treat cancer. Compounds may be used in methods for earlydetection of neoplastic transformation within the upper GI-tract as aligand to identify GCC expression. Similarly, screening, diagnosis,staging and post-operative surveillance of colorectal tumors may beundertaken using compounds which serve as a ligand to identify GCCexpression. The compounds may be used in diagnostic and therapeutictargeting of primary and metastatic esophageal and gastric tumors andmetastatic colorectal tumors as a ligand for GCCm and to facilitateintestinal recovery from insult e.g. intestinal adaptation, wherein theepithelium requires regeneration including, but not limited to, ischemicinsult, chemical insult, chemotherapy, trauma, and surgery.

In Vitro Diagnostics

According to some embodiments of the invention, compositions, kits andin vitro methods are provided for screening, diagnosing and analyzingpatients and patient samples to detect evidence of GCC expression bycells outside of the intestinal tract wherein the expression of GCC maybe suggestive of metastasized colorectal cancer or primary or metastaticstomach or esophageal cancer. In patients suspected of havingmetastasized colorectal cancer or primary or metastatic stomach oresophageal cancer evidence of GCC expression by cells outside of theintestinal tract is indicative of metastasized colorectal cancer orprimary or metastatic stomach or esophageal cancer and can be used inthe diagnosis, monitoring and staging of such patients. Furthermore, thepresent invention relates to methods, compositions and kits useful inthe in vitro screening, and analysis of patient and patient samples todetect evidence of GCC expression by tumor cells outside of theintestinal tract wherein the presence of cells that express GCC suggestsor confirms that a tumor is of colorectal or stomach or esophagealcancer origin. In an additional aspect of the invention, compositions,kits and methods are provided which are useful to visualize metastasizedcolorectal cancer or primary or metastatic stomach or esophageal cancercells.

In vitro screening and diagnostic compositions, methods and kits can beused in the monitoring of individuals who are in high risk groups forcolorectal, stomach or esophageal cancer such as those who have beendiagnosed with localized disease and/or metastasized disease and/orthose who are genetically linked to the disease. In vitro screening anddiagnostic compositions, methods and kits can be used in the monitoringof individuals who are undergoing and/or have been treated for primarycolorectal, stomach or esophageal cancer to determine if the cancer hasmetastasized. In vitro screening and diagnostic compositions, methodsand kits can be used in the monitoring of individuals who are undergoingand/or have been treated for colorectal, stomach or esophageal cancer todetermine if the cancer has been eliminated. In vitro screening anddiagnostic compositions, methods and kits can be used in the monitoringof individuals who are otherwise susceptible, i.e. individuals who havebeen identified as genetically predisposed such as by genetic screeningand/or family histories. Advancements in the understanding of geneticsand developments in technology as well as epidemiology allow for thedetermination of probability and risk assessment an individual has fordeveloping stomach or esophageal cancer. Using family health historiesand/or genetic screening, it is possible to estimate the probabilitythat a particular individual has for developing certain types of cancerincluding colorectal, stomach or esophageal cancer. Those individualsthat have been identified as being predisposed to developing aparticular form of cancer can be monitored or screened to detectevidence of colorectal, stomach or esophageal cancer. Upon discovery ofsuch evidence, early treatment can be undertaken to combat the disease.Accordingly, individuals who are at risk for developing colorectal,stomach or esophageal cancer may be identified and samples may beisolated form such individuals. The invention is particularly useful formonitoring individuals who have been identified as having family medicalhistories which include relatives who have suffered from colorectal,stomach or esophageal cancer. Likewise, the invention is particularlyuseful to monitor individuals who have been diagnosed as havingcolorectal, stomach or esophageal cancer and, particularly those whohave been treated and had tumors removed and/or are otherwiseexperiencing remission including those who have been treated forcolorectal, stomach or esophageal cancer.

In vitro screening and diagnostic compositions, methods and kits can beused in the analysis of tumors. Expression of GCC is a marker for celltype and suggests the origin of adenocarcinoma of unconfirmed origin maybe colorectal, stomach or esophageal tumors. Detection of GCC expressioncan also be used to assist in an initial diagnosis of colorectal,stomach or esophageal cancer or to confirm such diagnosis. Tumorsbelieved to be colorectal, stomach or esophageal in origin can beconfirmed as such using the compositions, methods and kits of theinvention.

In vitro screening and diagnostic compositions, kits and methods of theinvention can be used to analyze tissue samples from the stomach oresophagus to identify primary stomach or esophageal cancer.

In vitro screening and diagnostic compositions, kits and methods of theinvention can be used to analyze tissue samples from the colon to detectthe amount of invasion by primary colorectal cancer into the intestinaltissue.

According to the invention, compounds are provided which bind to GCCprotein. Normal tissue in the body does not have GCC protein exceptcells of the intestinal tract. The expression of GCC is a marker forcell type and is useful in the identification of colorectal, stomach oresophageal cancer in extra-intestinal samples.

In some embodiments of the invention, non-colorectal tissue and fluidsamples or tumor samples may be screened to identify the presence orabsence of GCC protein. The presence of GCC in non-colorectal tissue andfluid samples or on cells from non-colorectal tissue samples suggestspossible stomach or esophageal cancer. The presence of GCC in a tumorsample or on tumor cells suggests that the tumor may be colorectal,stomach or esophageal in origin.

Samples may be obtained from resected tissue or biopsy materialincluding needle biopsy. Tissue section preparation for surgicalpathology may be frozen and prepared using standard techniques. Bindingassays on tissue sections are performed in fixed cells. It is alsocontemplated that tumor samples in body fluids such as blood, urine,lymph fluid, cerebral spinal fluid, amniotic fluid, vaginal fluid, semenand stool samples may also be screened to determine if such tumors arecolorectal, stomach or espophageal in origin.

Non-colorectal tissue samples may be obtained from any tissue exceptthose of the colorectal tract, i.e. the intestinal tract below the smallintestine (i.e. the large intestine (colon), including the cecum,ascending colon, transverse colon, descending colon, and sigmoid colon,and rectum) and additionally the duodenum and small intestine (jejunumand ileum). The normal cells of all tissue except those of thecolorectal tract do not express GCC. Thus if GCC protein are detected innon-colorectal samples, the possible presence of colorectal, stomach oresophageal cancer cells is suggested. In some preferred embodiments, thetissue samples are lymph nodes.

Tissue samples may be obtained by standard surgical techniques includinguse of biopsy needles. One skilled in the art would readily appreciatethe variety of test samples that may be examined for GCC and recognizemethods of obtaining tissue samples.

Examples of body fluid samples include blood, urine, lymph fluid,cerebral spinal fluid, amniotic fluid, vaginal fluid and semen. In somepreferred embodiments, blood is used as a sample of body fluid. Cellsmay be isolated from fluid sample such as centrifugation. One skilled inthe art would readily appreciate the variety of test samples that may beexamined for GCC. Test samples may be obtained by such methods aswithdrawing fluid with a syringe or by a swab. One skilled in the artwould readily recognize other methods of obtaining test samples.

In an assay using a blood sample, the blood plasma may be separated fromthe blood cells. The blood plasma may be screened for GCC includingtruncated proteins which are released into the blood when one or moreGCC are cleaved from or sloughed off from tumor cells. In someembodiments, blood cell fractions are screened for the presence ofcolorectal, stomach or esophageal tumor cells. In some embodiments,lymphocytes present in the blood cell fraction are screened by lysingthe cells and detecting the presence of GCC protein which may be presentas a result of the presence of any stomach or esophageal tumor cellsthat may have been engulfed by the blood cell. In some preferredembodiments.

The present invention provides methods f detecting the presence ofdetecting presence of GCC in sample using compounds of the invention asa detectable ligand of GCC. The compounds of the invention used in thisassay may be used as research reagents as well as diagnostics sinceconsiderable laboratory research is conducted in which GCC presence mustbe detected. The GCC binding assay uses a detectable compounds to bindto any GCC present and thus indicate the presence of the receptor in asample.

The present invention also relates to methods of identifying individualssuffering from colorectal, stomach or esophageal cancer by detectingpresence of GCC in sample of tumor.

The GCC binding assay can be readily performed by those having ordinaryskill in the art using readily available starting materials. GCC bindingassays may be performed a variety of ways but each essentially identifywhether or not GCC protein is present in a sample by determining whetheror not a detectable compound binds to a receptor in a sample. Briefly,the assay consists of incubating a sample with a constant concentrationof compound of the invention such as 1×10⁻¹⁰ M to 5×10⁻¹⁰ M of¹²⁵I-labeled compound. As a control, a duplicate preparation of a sampleknown to contain GCC are incubated with a duplicate concentration of¹²⁵I-labeled compound of the invention. Assays are incubated toequilibrium (for example 2 hours) and the sample is analyzed todetermine whether or not ¹²⁵I-compound of the invention is bound tomaterial in the sample. The . ¹²⁵I-compound of the invention/sample ispassed through a filter which is capable of allowing .sup. ¹²⁵I-compoundof the invention to pass through but not capable of allowing GCC to passthrough. Thus, if GCC is present in the sample, it will bind the¹²⁵I-compound of the invention which will then be trapped by the filter.Detection of . ¹²⁵]-compound of the invention in the filter indicatesthe presence of GCC in the sample. In some preferred embodiments, thefilter is Whitman GFB glass filter paper. Controls include using sampleswhich are known to contain GCC, e.g. intestinal membranes from ratintestine, human intestine, T84 cells, isolated GCC protein or cellsexpressing cloned nucleotide sequence encoding GCC.

The compounds of the invention may be prepared routinely by any of thefollowing known techniques. In addition to being conjugated to ¹²⁵I,compounds of the invention may be detectable by binding it to otherradionuclides such as: ⁴³K, ⁵²Fe, ⁵⁷Co, ⁶⁷Cu, ⁶⁷Ga, ⁶⁸Ga, ⁷⁷Br,⁸¹Rb/⁸¹MKr, ⁸⁷MSr, ⁹⁹MTc, ¹¹¹In, ¹¹³MIn, ¹²³I, ¹²⁵I, ¹²⁷Cs, ¹²⁹Cs, ¹³¹I,¹³²I, ¹⁹⁷Hg, ²⁰³Pb and ²⁰⁶206Bi, ⁴⁷Sc, ⁶⁷Cu, ⁹⁰Y, ¹⁰⁹Pd, ¹²³I, ¹²⁵I,¹³¹I, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁹⁹Au, ²¹¹At, ²¹²Pb and ²²Bi, ³²P and ³³P ⁷¹Ge,⁷⁷As, ¹⁰³Pb, ¹⁰⁵Rh, ¹¹¹Ag, ¹¹⁹Sb, ¹²¹Sn, ¹³¹Cs, ¹⁴³Pr, ¹⁶¹T, ¹⁷⁷Lu,¹⁹¹Os, ¹⁹³MPt, ¹⁹⁷Hg, as well as all beta negative and/or auger emittersor by binding it to other labels such as fluorescein or enzymes. Each ofthe labeling means described above for detectably labeling antibodiescan be adapted to label compounds of the invention and are considered tobe described as such herein.

The compound of the invention may be detectably labeled is by linking itto an enzyme. The enzyme, when subsequently exposed to its substrate,reacts with the substrate and generates a chemical moiety which can bedetected, for example, by spectrophotometric, fluorometric or visualmeans. Enzymes which can be used to detectably label antibodies include,but are not limited to malate dehydrogenase, staphylococcal nuclease,delta-5-steroid isomerase, yeast alcohol dehydrogenase,alpha-glycerophosphate dehydrogenase, triose phosphate isomerase,horseradish peroxidase, alkaline phosphatase, asparaginase, glucoseoxidase, beta-galactosidase, ribonuclease, urease, catalase,glucose-6-phosphate dehydrogenase, glucoamylase andacetylcholinesterase. One skilled in the art would readily recognizeother enzymes which may also be used. It is also possible to label thecompound with a fluorescent compound. When the fluorescent-labeledantibody is exposed to light of the proper wave length, its presence canbe detected due to its fluorescence. Among the most commonly usedfluorescent labeling compounds are fluorescein isothiocyanate,rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehydeand fluorescamine. One skilled in the art would readily recognize otherfluorescent compounds which may also be used. Compounds of the inventioncan also be detectably labeled using fluorescence-emitting metals suchas ¹⁵²Eu, or others of the lanthanide series. These metals can beattached to the protein-specific antibody using such metal chelatinggroups as diethylenetriaminepentaacet-ic acid (DTPA) orethylenediamine-tetraacetic acid (EDTA). One skilled in the art wouldreadily recognize other fluorescence-emitting metals as well as othermetal chelating groups which may also be used. Compounds of theinventions can also be detectably labeled by coupling to achemiluminescent compound. The presence of the chemiluminescent-labeledantibody is determined by detecting the presence of luminescence thatarises during the course of a chemical reaction. Examples ofparticularly useful chemoluminescent labeling compounds are luminol,isoluminol, theromatic acridinium ester, imidazole, acridinium salt andoxalate ester. One skilled in the art would readily recognize otherchemiluminescent compounds which may also be used. Likewise, abioluminescent compound may be used to label antibodies. Bioluminescenceis a type of chemiluminescence found in biological systems in which acatalytic protein increases the efficiency of the chemiluminescentreaction. The presence of a bioluminescent protein is determined bydetecting the presence of luminescence. Important bioluminescentcompounds for purposes of labeling are luciferin, luciferase andaequorin. One skilled in the art would readily recognize otherbioluminescent compounds which may also be used.

Detection of the compound of the invention may be accomplished by ascintillation counter if, for example, the detectable label is aradioactive gamma emitter. Alternatively, detection may be accomplishedby a fluorometer if, for example, the label is a fluorescent material.In the case of an enzyme label, the detection can be accomplished bycolorometric methods which employ a substrate for the enzyme. Detectionmay also be accomplished by visual comparison of the extent of enzymaticreaction of a substrate in comparison with similarly prepared standards.One skilled in the art would readily recognize other appropriate methodsof detection which may also be used.

Positive and negative controls may be performed in which known amountsof GCC protein and no GCC protein, respectively, are added to assaysbeing performed in parallel with the test assay. One skilled in the artwould have the necessary knowledge to perform the appropriate controls.In addition, the kit may comprise instructions for performing the assay.Additionally the kit may optionally comprise depictions or photographsthat represent the appearance of positive and negative results.

Anti-compound of the invention antibodies may be generated, detectablylabeled in a manner described above for labeling compound of theinvention, and used to detect compound of the invention bound to GCC inthe sample. Anti-compound of the invention antibodies may also bedesigned for immuno-PCR, immuno-RNA amplification, magnetic sorting etc.

Kits include containers comprising detectable compound of the inventionstogether with containers having positive and/or negative controls, i.e.samples which contain GCC and samples which contain no GCC,respectively. The detectable compound of the inventions is preferablylabeled. Additional components in some kits include solid support,buffer, and instructions for carrying out the assay. Additionally thekit may optionally comprise depictions or photographs that represent theappearance of positive and negative results.

The GCC binding assay is useful for detecting GCC in homogenized tissuesamples and body fluid samples including the plasma portion.

In Vivo Imaging and Therapeutics

According to some embodiments of the invention, compositions and in vivomethods are provided for detecting, imaging, or treating metastaticcolorectal cancer and primary and/or metastatic stomach or esophagealtumors in an individual.

When the conjugated compositions of the present invention areadministered outside the intestinal tract such as when administered inthe circulatory system, they remain segregated from the cells that linethe intestinal tract and will bind only to cells outside the intestinaltract which express GCC. The conjugated compositions will not bind tothe normal cells but will bind to metastatic colorectal cancer cells andprimary and/or metastatic stomach or esophageal cells. Thus, the activemoieties of conjugated compositions administered outside the intestinaltract are delivered to cells which express GCC such as metastaticcolorectal cancer cells and primary and/or metastatic stomach oresophageal cancer cells.

Therapeutic and diagnostic pharmaceutical compositions useful in thepresent invention include conjugated compounds that specifically targetcells that express GCC. These conjugated compounds include moieties thatbind to GCC which do not bind to cells of normal tissue in the bodyexcept cells of the intestinal tract since the cells of other tissues donot express GCC.

Unlike normal colorectal cells, cancer cells that express GCC areaccessible to substances administered outside the intestinal tract, forexample administered in the circulatory system. The only GCC in normaltissue exist in the apical membranes of intestinal mucosa cells and thuseffectively isolated from the targeted cancer chemotherapeutics andimaging agents administered outside the intestinal tract by theintestinal mucosa barrier. Thus, metastatic colorectal cancer andprimary and/or metastatic stomach or esophageal cancer cells may betargeted by conjugated compounds of the present invention by introducingsuch compounds outside the intestinal tract such as for example byadministering pharmaceutical compositions that comprise conjugatedcompounds into the circulatory system.

One having ordinary skill in the art can identify individuals suspectedof suffering from metastatic colorectal cancer and primary and/ormetastatic stomach or esophageal cancer. In those individuals diagnosedwith colorectal, stomach or esophageal cancer, it is not unusual and insome cases standard therapy to suspect metastasis and aggressivelyattempt to eradicate metastasized cells. The present invention providespharmaceutical compositions and methods for imaging and thereby willmore definitively diagnose primary and metastastic disease. Further, thepresent invention provides pharmaceutical compositions comprisingtherapeutic agents and methods for specifically targeting andeliminating metastatic colorectal cancer and primary and/or metastaticstomach or esophageal cancer cells. Further, the present inventionprovides pharmaceutical compositions that comprise therapeutics andmethods for specifically eliminating metastatic colorectal cancer andprimary and/or metastatic stomach or esophageal cancer cells.

The pharmaceutical compositions which comprise conjugated compositionsof the present invention may be used to diagnose or treat individualssuffering from metastatic colorectal cancer and primary and/ormetastatic stomach or esophageal tumors.

The present invention relies upon the use of a compounds of theinvention in a conjugated composition. The compounds of the inventionare essentially a portion of the conjugated composition which acts as aligand to GCCI and thus specifically binds to it. The conjugatedcomposition also includes an active moiety which is associated with thecompounds of the invention; the active moiety being an active agentwhich is either useful to image, target, neutralize or kill the cell.

According to some aspects of the present invention, compounds comprisethe compound of the invention linked to an active moiety that may be atherapeutic agent or an imaging agent. One having ordinary skill in theart can readily recognize the advantages of being able to specificallytarget cancer cells with a compound of the invention and conjugate suchcompound of the invention with many different active agents.

Chemotherapeutics useful as active moieties which when conjugated to acompound of the invention are specifically delivered to cells thatexpress GCC such as metastatic colorectal cancer cells, stomach cancercells or esophageal cancer cells, are typically small chemical entitiesproduced by chemical synthesis. Chemotherapeutics include cytotoxic andcytostatic drugs. Chemotherapeutics may include those which have othereffects on cells such as reversal of the transformed state to adifferentiated state or those which inhibit cell replication. Examplesof chemotherapeutics include common cytotoxic or cytostatic drugs suchas for example: methotrexate (amethopterin), doxorubicin (adrimycin),daunorubicin, cytosinarabinoside, etoposide, 5-4 fluorouracil,melphalan, chlorambucil, and other nitrogen mustards (e.g.cyclophosphamide), cis-platinum, vindesine (and other vinca alkaloids),mitomycin and bleomycin. Other chemotherapeutics include: purothionin(barley flour oligopeptide), macromomycin. 1,4-benzoquinone derivativesand trenimon.

Toxins are useful as active moieties. When a toxin is conjugated to acompound of the invention, the conjugated composition is specificallydelivered to a cell that expresses GCC such as metastatic colorectalcancer, stomach cancer or esophageal cancer cells by way of the compoundof the invention and the toxin moiety kills the cell. Toxins aregenerally complex toxic products of various organisms includingbacteria, plants, etc. Examples of toxins include but are not limitedto: ricin, ricin A chain (ricin toxin), Pseudomonas exotoxin (PE),diphtheria toxin (DT), Clostridium perfringens phospholipase C (PLC),bovine pancreatic ribonuclease (BPR), pokeweed antiviral protein (PAP),abrin, abrin A chain (abrin toxin), cobra venom factor (CVF), gelonin(GEL), saporin (SAP), modeccin, viscumin and volkensin. As discussedabove, when protein toxins are employed with compound of the inventionpeptides, conjugated compositions may be produced using recombinant DNAtechniques. Briefly, a recombinant DNA molecule can be constructed whichencodes both the compound and the toxin on a chimeric gene. When thechimeric gene is expressed, a fusion protein is produced which includesa compound of the invention and an active moiety. Protein toxins arealso useful to form conjugated compounds with compound of the inventionthrough non-peptidyl bonds.

In addition, there are other approaches for utilizing active agents forthe treatment of cancer. For example, conjugated compositions may beproduced which include a compound of the invention and an active moietywhich is an active enzyme. The compound of the invention specificallylocalizes the conjugated composition to the tumor cells. An inactiveprodrug which can be converted by the enzyme into an active drug isadministered to the patient. The prodrug is only converted to an activedrug by the enzyme which is localized to the tumor. An example of anenzyme/prodrug pair includes alkaline phosphatase/etoposidephosphate. Insuch a case, the alkaline phosphatase is conjugated to a compound of theinvention. The conjugated compound is administered and localizes at thecancer cell. Upon contact with etoposidephosphate (the prodrug), theetoposidephosphate is converted to etoposide, a chemotherapeutic drugwhich is taken up by the cancer cell. Radiosensitizing agents aresubstances that increase the sensitivity of cells to radiation. Examplesof radiosensitizing agents include nitroimidazoles, metronidazole andmisonidazole (see: DeVita, V. T. Jr. in Harrison's Principles ofInternal Medicine, p. 68, McGraw-Hill Book Co., N.Y. 1983, which isincorporated herein by reference). The conjugated compound thatcomprises a radiosensitizing agent as the active moiety is administeredand localizes at the metastatic colorectal cancer cell and primaryand/or metastatic stomach or esophageal cancer cell. Upon exposure ofthe individual to radiation, the radiosensitizing agent is “excited” andcauses the death of the cell.

Radionuclides may be used in pharmaceutical compositions that are usefulfor radiotherapy or imaging procedures. Examples of radionuclides usefulas toxins in radiation therapy include: ⁴⁷Sc, ⁶⁷Cu, ⁹⁰Y, ¹⁰⁹Pd, ¹²³I,¹²⁵I, ¹³¹I, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁹⁹Au, ²¹¹At, ²¹²Pb and ²¹²Bi. Otherradionuclides which have been used by those having ordinary skill in theart include: ³²P and ³³P, ⁷¹Ge, ⁷⁷As, ¹⁰³Pb, ¹⁰⁵Rh, ¹¹¹Ag, ¹¹⁹Sb, ¹²¹Sn,¹³¹Cs, ¹⁴³Pr, ¹⁶¹Tb, ¹⁷⁷Lu, ¹⁹¹Os, ¹⁹³MPt, ¹⁹⁷Hg, all beta negativeand/or auger emitters. Some preferred radionuclides include: ⁹⁰Y, ¹³¹I,²¹¹At and ²¹²Pb/²¹²Bi.

According to the present invention, the active moieties may be animaging agent. Imaging agents are useful diagnostic procedures as wellas the procedures used to identify the location of cancer cells. Imagingcan be performed by many procedures well-known to those having ordinaryskill in the art and the appropriate imaging agent useful in suchprocedures may be conjugated to a compound of the invention bywell-known means. Imaging can be performed, for example, byradioscintigraphy, nuclear magnetic resonance imaging (MRI) or computedtomography (CT scan). The most commonly employed radionuclide imagingagents include radioactive iodine and indium. Imaging by CT scan mayemploy a heavy metal such as iron chelates. MRI scanning may employchelates of gadolinium or manganese. Additionally, positron emissiontomography (PET) may be possible using positron emitters of oxygen,nitrogen, iron, carbon, or gallium. Example of radionuclides useful inimaging procedures include: ⁴³K, ⁵²Fe, ⁵⁷Co, ⁶⁷Cu, ⁶⁷Ga, ⁶⁸Ga, ⁷⁷Br,⁸¹Rb/⁸¹MKr, ⁸⁷MSr, ⁹⁹MTc, ¹¹¹In, ¹¹³MIn, ¹²³I, ¹²⁵I, ¹²⁷Cs, ¹²⁹Cs, ¹³¹I,¹³²I, ¹⁹⁷Hg, ²⁰³Pb and ²⁰⁶206Bi.

It is preferred that the conjugated compositions be non-immunogenic orimmunogenic at a very low level. Accordingly, it is preferred that thecompound of the invention be a small, poorly immunogenic ornon-immunogenic peptide or a non-peptide.

Compounds of the inventions are conjugated to active agents by a varietyof well-known techniques readily performed without undue experimentationby those having ordinary skill in the art. The technique used toconjugate the compound of the invention to the active agent is dependentupon the molecular nature of the compound of the invention and theactive agent. After the compound of the invention and the active agentare conjugated to form a single molecule, assays may be performed toensure that the conjugated molecule retains the activities of themoieties. The competitive binding assay described above may be used toconfirm that the compound of the invention retains its binding activityas a conjugated compound. Similarly, the activity of the active moietymay be tested using various assays for each respective type of activeagent. Radionuclides retain there activity, i.e. their radioactivity,irrespective of conjugation. With respect to active agents which aretoxins, drugs and targeting agents, standard assays to demonstrate theactivity of unconjugated forms of these compounds may be used to confirmthat the activity has been retained.

Conjugation may be accomplished directly between the compound of theinvention and the active agent or linking, intermediate molecular groupsmay be provided between the compound of the invention and the activeagent. Linkers are particularly useful to facilitate conjugation byproviding attachment sites for each moiety. Linkers may includeadditional molecular groups which serve as spacers to separate themoieties from each other to prevent either from interfering with theactivity of the other.

One having ordinary skill in the art may conjugate a compound of theinvention to a chemotherapeutic drug using well-known techniques. Forexample, Magerstadt, M. Antibody Conjugates and Malignant Disease.(1991) CRC Press, Boca Raton, USA, pp. 110-152) which is incorporatedherein by reference, teaches the conjugation of various cytostatic drugsto amino acids of antibodies. Such reactions may be applied to conjugatechemotherapeutic drugs to compound of the inventions with an appropriatelinker. Most of the chemotherapeutic agents currently in use in treatingcancer possess functional groups that are amenable to chemical linkingdirectly with proteins. For example, free amino groups are available onmethotrexate, doxorubicin, daunorubicin, cytosinarabinoside, cis-platin,vindesine, mitomycin and bleomycin while free carboxylic acid groups areavailable on methotrexate, melphalan, and chlorambucil. These functionalgroups, that is free amino and carboxylic acids, are targets for avariety of homobifunctional and heterobifunctional chemical linkingagents which can link these drugs directly to the single free aminogroup of an antibody. For example, one procedure for linking compound ofthe inventions which have a free amino group to active agents which havea free amino group such as methotrexate, doxorubicin, daunorubicin,cytosinarabinoside, cis-platin, vindesine, mitomycin and bleomycin, oralkaline phosphatase, or protein- or peptide-based toxin employshomobifunctional succinimidyl esters, preferably with carbon chainspacers such as disuccinimidyl suberate (Pierce Co, Rockford, Ill.). Inthe event that a cleavable conjugated compound is required, the sameprotocol would be employed utilizing 3,3′-dithiobis(sulfosuccinimidylpropionate; Pierce Co.).

In order to conjugate a compound of the invention that is a peptide orprotein to a peptide-based active agent such as a toxin, the compound ofthe invention and the toxin may be produced as a single, fusion proteineither by standard peptide synthesis or recombinant DNA technology, bothof which can be routinely performed by those having ordinary skill inthe art. Alternatively, two peptides, the compound of the invention andthe peptide-based toxin may be produced and/or isolated as separatepeptides and conjugated using linkers. As with conjugated compositionsthat contain chemotherapeutic drugs, conjugation of compound of theinvention and toxins can exploit the ability to modify the single freeamino group of a compound of the invention while preserving thereceptor-binding function of this molecule.

One having ordinary skill in the art may conjugate a compound of theinvention to a radionuclide using well-known techniques. For example,Magerstadt, M. (1991) Antibody Conjugates And Malignant Disease, CRCPress, Boca Raton, Fla.; and Barchel, S. W. and Rhodes, B. H., (1983)Radioimaging and Radiotherapy, Elsevier, NY N.Y., each of which isincorporated herein by reference, teach the conjugation of varioustherapeutic and diagnostic radionuclides to amino acids of antibodies.

The present invention provides pharmaceutical compositions that comprisethe conjugated compounds of the invention and pharmaceuticallyacceptable carriers or diluents. The pharmaceutical composition of thepresent invention may be formulated by one having ordinary skill in theart. Suitable pharmaceutical carriers are described in Remington'sPharmaceutical Sciences, A. Osol, a standard reference text in thisfield, which is incorporated herein by reference. In carrying outmethods of the present invention, conjugated compounds of the presentinvention can be used alone or in combination with other diagnostic,therapeutic or additional agents. Such additional agents includeexcipients such as coloring, stabilizing agents, osmotic agents andantibacterial agents. Pharmaceutical compositions are preferably sterileand pyrogen free.

The conjugated compositions of the invention can be, for example,formulated as a solution, suspension or emulsion in association with apharmaceutically acceptable parenteral vehicle. Examples of suchvehicles are water, saline, Ringer's solution, dextrose solution, and 5%human serum albumin. Liposomes may also be used. The vehicle may containadditives that maintain isotonicity (e.g., sodium chloride, mannitol)and chemical stability (e.g., buffers and preservatives). Theformulation is sterilized by commonly used techniques. For example, aparenteral composition suitable for administration by injection isprepared by dissolving 1.5% by weight of active ingredient in 0.9%sodium chloride solution.

The pharmaceutical compositions according to the present invention maybe administered as either a single dose or in multiple doses. Thepharmaceutical compositions of the present invention may be administeredeither as individual therapeutic agents or in combination with othertherapeutic agents. The treatments of the present invention may becombined with conventional therapies, which may be administeredsequentially or simultaneously.

The pharmaceutical compositions of the present invention may beadministered by any means that enables the conjugated composition toreach the targeted cells. In some embodiments, routes of administrationinclude those selected from the group consisting of intravenous,intraarterial, intraperitoneal, local administration into the bloodsupply of the organ in which the tumor resides or directly into thetumor itself. In addition to an intraoperative spray, conjuagatedcompounds may be delivered intrathecally, intraventrically,stereotactically, intrahepatically such as via the portal vein, byinhalation, and intrapleurally. Intravenous administration is thepreferred mode of administration. It may be accomplished with the aid ofan infusion pump.

The dosage administered varies depending upon factors such as: thenature of the active moiety; the nature of the conjugated composition;pharmacodynamic characteristics; its mode and route of administration;age, health, and weight of the recipient; nature and extent of symptoms;kind of concurrent treatment; and frequency of treatment.

Because conjugated compounds are specifically targeted to cells with oneor more GCC molecules, conjugated compounds which comprisechemotherapeutics or toxins are administered in doses less than thosewhich are used when the chemotherapeutics or toxins are administered asunconjugated active agents, preferably in doses that contain up to 100times less active agent. In some embodiments, conjugated compounds whichcomprise chemotherapeutics or toxins are administered in doses thatcontain 10-100 times less active agent as an active moiety than thedosage of chemotherapeutics or toxins administered as unconjugatedactive agents. To determine the appropriate dose, the amount of compoundis preferably measured in moles instead of by weight. In that way, thevariable weight of different compounds of the invention does not affectthe calculation. Presuming a one to one ratio of compound of theinvention to active moiety in conjugated compositions of the invention,less moles of conjugated compounds may be administered as compared tothe moles of unconjugated compounds administered, preferably up to 100times less moles.

Typically, chemotherapeutic conjugates are administered intravenously inmultiple divided doses.

Up to 20 gm IV/dose of methotrexate is typically administered in anunconjugated form. When methotrexate is administered as the activemoiety in a conjugated compound of the invention, there is a 10- to100-fold dose reduction. Thus, presuming each conjugated compoundincludes one molecule of methotrexate conjugated to one compound of theinvention, of the total amount of conjugated compound administered, upto about 0.2-2.0 g of methotrexate is present and thereforeadministered. In some embodiments, of the total amount of conjugatedcompound administered, up to about 200 mg-2 g of methotrexate is presentand therefore administered.

To dose conjugated compositions comprising compounds of the inventionlinked to active moieties that are radioisotopes in pharmaceuticalcompositions useful as imaging agents, it is presumed that each compoundof the invention is linked to one radioactive active moiety. The amountof radioisotope to be administered is dependent upon the radioisotope.Those having ordinary skill in the art can readily formulate the amountof conjugated compound to be administered based upon the specificactivity and energy of a given radionuclide used as an active moiety.Typically 0.1-100 millicuries per dose of imaging agent, preferably 1-10millicuries, most often 2-5 millicuries are administered. Thus,pharmaceutical compositions according to the present invention useful asimaging agents which comprise conjugated compositions comprising acompound of the invention and a radioactive moiety comprise 0.1-100millicuries, in some embodiments preferably 1-10 millicuries, in someembodiments preferably 2-5 millicuries, in some embodiments morepreferably 1-5 millicuries. Examples of dosages include: ¹³¹I-betweenabout 0.1-100 millicuries per dose, in some embodiments preferably 1-10millicuries, in some embodiments 2-5 millicuries, and in someembodiments about 4 millicuries; . ¹¹¹In between about 0.1-100millicuries per dose, in some embodiments preferably 1-10 millicuries,in some embodiments 1-5 millicuries, and in some embodiments about 2millicuries; . ⁹⁹ mTc-between about 0.1-100 millicuries per dose, insome embodiments preferably 5-75 millicuries, in some embodiments 10-50millicuries, and in some embodiments about 27 millicuries. Wessels B. W.and R. D. Rogus (1984) Med. Phys. 11:638 and Kwok, C. S. et al. (1985)Med. Phys. 12:405, both of which are incorporated herein by reference,disclose detailed dose calculations for diagnostic and therapeuticconjugates which may be used in the preparation of pharmaceuticalcompositions of the present invention which include radioactiveconjugated compounds.

One aspect of the present invention relates to a method of treatingindividuals suspected of suffering from metastatic colorectal cancer andprimary and/or metastatic stomach or esophageal cancer. Such individualsmay be treated by administering to the individual a pharmaceuticalcomposition that comprises a pharmaceutically acceptable carrier ordiluent and a conjugated compound that comprises a compound of theinvention and an active moiety wherein the active moiety is aradiostable therapeutic agent. In some embodiments of the presentinvention, the pharmaceutical composition comprises a pharmaceuticallyacceptable carrier or diluent and a conjugated compound that comprises acompound of the invention and an active moiety wherein the active moietyis a radiostable therapeutic agent. In some embodiments of the presentinvention, the pharmaceutical composition comprises a pharmaceuticallyacceptable carrier or diluent and a conjugated compound that comprises acompound of the invention and an active moiety wherein the active moietyis a radiostable active agent selected from the group consisting of:methotrexate, doxorubicin, daunorubicin, cytosinarabinoside, etoposide,5-4 fluorouracil, melphalan, chlorambucil, cis-platinum, vindesine,mitomycin, bleomycin, purothionin, macromomycin, 1,4-benzoquinonederivatives, trenimon, ricin, ricin A chain, Pseudomonas exotoxin,diphtheria toxin, Clostridium perfringens phospholipase C, bovinepancreatic ribonuclease, pokeweed antiviral protein, abrin, abrin Achain, cobra venom factor, gelonin, saporin, modeccin, viscumin,volkensin, alkaline phosphatase, nitroimidazole, metronidazole andmisonidazole. The individual being treated may be diagnosed as havingmetastasized colorectal, stomach or esophageal cancer or may bediagnosed as having primary colorectal, stomach or esophageal cancer andmay undergo the treatment proactively in the event that there is somemetastasis as yet undetected. The pharmaceutical composition contains atherapeutically effective amount of the conjugated composition. Atherapeutically effective amount is an amount which is effective tocause a cytotoxic or cytostatic effect on cancer cells without causinglethal side effects on the individual.

One aspect of the present invention relates to a method of treatingindividuals suspected of suffering from metastatic colorectal cancer andprimary and/or metastatic stomach or esophageal cancer. Such individualsmay be treated by administering to the individual a pharmaceuticalcomposition that comprises a pharmaceutically acceptable carrier ordiluent and a conjugated compound that comprises a compound of theinvention and an active moiety wherein the active moiety is aradioactive. In some embodiments of the present invention, thepharmaceutical composition comprises a pharmaceutically acceptablecarrier or diluent and a conjugated compound that comprises a compoundof the invention and an active moiety wherein the active moiety is aradioactive and the compound of the invention is an antibody. In someembodiments of the present invention, the pharmaceutical compositioncomprises a pharmaceutically acceptable carrier or diluent and aconjugated compound that comprises a compound of the invention and anactive moiety wherein the active moiety is a radioactive agent selectedfrom the group consisting of: ⁴⁷Sc, ⁶⁷Cu, ⁹⁰Y, ¹⁰⁹Pd, ¹²³I, ¹²⁵I, ¹³¹I,¹⁸⁶Re, ¹⁸⁸Re, ¹⁹⁹Au, ²¹¹At, ²¹²Pb and ²¹²Bi, ³²P and ³³P, ⁷¹Ge, ⁷⁷As,¹⁰³Pb, ¹⁰⁵Rh, ¹¹¹Ag, ¹¹⁹Sb, ¹²¹Sn, ¹³¹Cs, ¹⁴³Pr, ¹⁶¹Tb, ¹⁷⁷Lu, ¹⁹¹Os,¹⁹³ MPt, ¹⁹⁷Hg, and all beta negative and/or auger emitters. Somepreferred radionuclides include: ⁹⁰Y, ¹³¹I, ²¹¹At and ²¹²Pb/²¹²Bi. allbeta negative and/or auger emitters. The individual being treated may bediagnosed as having metastasized cancer or may be diagnosed as havinglocalized cancer and may undergo the treatment proactively in the eventthat there is some metastasis as yet undetected. The pharmaceuticalcomposition contains a therapeutically effective amount of theconjugated composition. A therapeutically effective amount is an amountwhich is effective to cause a cytotoxic or cytostatic effect onmetastatic colorectal cancer and primary and/or metastatic stomach oresophageal cancer cells without causing lethal side effects on theindividual. The composition may be injected intratumorally into primarytumors.

One aspect of the present invention relates to a method of detectingmetastatic colorectal cancer and primary and/or metastatic stomach oresophageal cancer cells in an individual suspected of suffering fromprimary or metastasized colorectal, stomach or esophageal cancer byradioimaging. Individuals may be suspected of having primary stomach oresophageal tumors which diagnosis can be confirmed by administering tothe individual, an imaging agent which binds to GCC. Tumors can beimaged by detecting localization at the stomach or esophagus.Individuals may be diagnosed as suffering from metastasized colorectal,stomach or esophageal cancer and the metastasized colorectal, stomach oresophageal cancer cells may be detected by administering to theindividual, preferably by intravenous administration, a pharmaceuticalcomposition that comprises a pharmaceutically acceptable carrier ordiluent and a conjugated compound that comprises a compound of theinvention and an active moiety wherein the active moiety is aradioactive and detecting the presence of a localized accumulation oraggregation of radioactivity, indicating the presence of cells with GCC.In some embodiments of the present invention, the pharmaceuticalcomposition comprises a pharmaceutically acceptable carrier or diluentand a conjugated compound that comprises a compound of the invention andan active moiety wherein the active moiety is a radioactive. In someembodiments of the present invention, the pharmaceutical compositioncomprises a pharmaceutically acceptable carrier or diluent and aconjugated compound that comprises a compound of the invention and anactive moiety wherein the active moiety is a radioactive agent selectedfrom the group consisting of: radioactive heavy metals such as ironchelates, radioactive chelates of gadolinium or manganese, positronemitters of oxygen, nitrogen, iron, carbon, or gallium, ⁴³K, ⁵²Fe, ⁵⁷Co,⁶⁷Cu, ⁶⁷Ga, ⁶⁸Ga, ⁷⁷Br, ⁸¹Rb/⁸¹r, ⁸⁷MSr, ⁹⁹MTc, ¹¹¹In, ¹¹³MIn, ¹²³I,¹²⁵I, ¹²⁷Cs, ¹²⁹Cs, ¹³¹I, ¹³²I, ¹⁹⁷Hg, ²⁰³Pb and ²⁰⁶206Bi. Theindividual being treated may be diagnosed as having metastasizingcolorectal, stomach or esophageal cancer or may be diagnosed as havinglocalized colorectal, stomach or esophageal cancer and may undergo thetreatment proactively in the event that there is some metastasis as yetundetected. The pharmaceutical composition contains a diagnosticallyeffective amount of the conjugated composition. A diagnosticallyeffective amount is an amount which can be detected at a site in thebody where cells with GCC are located without causing lethal sideeffects on the individual.

Photodynamic Imaging and Therapy

According to some embodiments of the invention, compounds of theinvention are conjugates to photoactivated imaging agents ortherapeutics. Maier A. et al. Lasers in Surgery and Medicine 26:461-466(2000) which is incorporated herein by reference disclose an example ofphotodynamic therapy. QLT, Inc (Vancouver, BC) commercially distributephotosensitive active agents which can be linked to a compound of theinvention. Such conjugated compounds can be used in photodynamictherapeutic and imaging protocols to activate the GCC-bound conjugatedagents which are thus targeted to tumor cells. In some embodiments, theconjugated compounds are applied as an intraoperative spray which issubsequently exposed to light to activate compounds bound to cells thatexpress GCC.

In some embodiments, the photodynamic agent is fluorophore orporphyrins. Examples of porphyrin include: hematoporphyrin derivative(HPD) and porfimer sodium (Photofrin.RTM.). A second generationphotosensitizers is BPD verteporfin. In some embodiments the fluorophoreis tetramethylrotamine. Lasers are generally the primary light sourceused to activate porphyrins. Light Emitting Diodes (LEDs) and florescentlight sources may also be used in some applications.

In addition to an intraoperative spray, conjuagated compounds may bedelivered intrathecally, intraventrically, stereotactically,intrahepatically such as via the portal vein, by inhalation, andintrapleurally.

Drug Delivery Targeted to Colorectal, Stomach or Esophageal Cancer CellsGenerally

Another aspect of the invention relates to unconjugated and conjugatedcompositions which comprise a compound of the invention used to delivertherapeutic agents to cells that comprise a GCC such as metastaticcolorectal cancer and primary and/or metastatic stomach or esophagealcancer cells. In some embodiments, the agent is a drug or toxin such as:methotrexate, doxorubicin, daunorubicin, cytosinarabinoside, etoposide,5-4 fluorouracil, melphalan, chlorambucil, cis-platinum, vindesine,mitomycin, bleomycin, purothionin, macromomycin, 1,4-benzoquinonederivatives, trenimon, ricin, ricin A chain, Pseudomonas exotoxin,diphtheria toxin, Clostridium perfringens phospholipase C, bovinepancreatic ribonuclease, pokeweed antiviral protein, abrin, abrin Achain, cobra venom factor, gelonin, saporin, modeccin, viscumin,volkensin, alkaline phosphatase, nitroimidazole, metronidazole andmisonidazole. Genetic material is delivered to cancer cells to producean antigen that can be targeted by the immune system or to produce aprotein which kills the cell or inhibits its proliferation. In someembodiments, a compound of the invention is used to deliver nucleicacids that encode nucleic acid molecules which replace defectiveendogenous genes or which encode therapeutic proteins. In someembodiments, the compositions are used in gene therapy protocols todeliver to individuals, genetic material needed and/or desired to makeup for a genetic deficiency.

In some embodiments, the compound of the invention is combined with orincorporated into a delivery vehicle thereby converting the deliveryvehicle into a specifically targeted delivery vehicle. For example, acompound of the invention may be integrated into the outer portion of aviral particle making such a virus a GCC-bearing cell specific virus.Similarly, the coat protein of a virus may be engineered such that it isproduced as a fusion protein which includes an active compound of theinvention peptide that is exposed or otherwise accessible on the outsideof the viral particle making such a virus a GCC-bearing cell-specificvirus. In some embodiments, a compound of the invention may beintegrated or otherwise incorporated into the liposomes wherein thecompound of the invention is exposed or otherwise accessible on theoutside of the liposome making such liposomes specifically targeted toGCC-bearing cells.

The active agent in the conjugated or unconjugated compositionsaccording to this aspect of the invention is a drug, toxin or nucleicacid molecule. The nucleic acid may be RNA or preferably DNA. In someembodiments, the nucleic acid molecule is an antisense molecule orencodes an antisense sequence whose presence in the cell inhibitsproduction of an undesirable protein. In some embodiments, the nucleicacid molecule encodes a ribozyme whose presence in the cell inhibitsproduction of an undesirable protein. In some embodiments, the nucleicacid molecule encodes a protein or peptide that is desirably produced inthe cell. In some embodiments, the nucleic acid molecule encodes afunctional copy of a gene that is defective in the targeted cell. Thenucleic acid molecule is preferably operably linked to regulatoryelements needed to express the coding sequence in the cell.

Liposomes are small vesicles composed of lipids. Genetic constructswhich encode proteins that are desired to be expressed in GCC-bearingcells are introduced into the center of these vesicles. The outer shellof these vesicles comprise a compound of the invention. LiposomesVolumes 1, 2 and 3 CRC Press Inc. Boca Raton Fla., which is incorporatedherein by reference, disclose preparation of liposome-encapsulatedactive agents which include antibodies in the outer shell. In thepresent invention, a compound of the invention is associated with the inthe outer shell. Unconjugated compositions which comprise a compound ofthe invention in the matrix of a liposome with an active agent inside.

In one embodiment, the delivery of normal copies of the p53 tumorsuppressor gene to the cancer cells is accomplished using a compound ofthe invention to target the gene therapeutic. Mutations of the p53 tumorsuppressor gene appears to play a prominent role in the development ofmany cancers. One approach to combating this disease is the delivery ofnormal copies of this gene to the cancer cells expressing mutant formsof this gene. Genetic constructs that comprise normal p53 tumorsuppressor genes are incorporated into liposomes that comprise acompound of the invention. The composition is delivered to the tumor.Compound of the inventions specifically target and direct the liposomescontaining the normal gene to correct the lesion created by mutation ofp53 suppressor gene. Preparation of genetic constructs is with the skillof those having ordinary skill in the art. The present invention allowssuch construct to be specifically targeted by using the compound of theinventions of the present invention. The compositions of the inventioninclude a compound of the invention associated with a delivery vehicleand a gene construct which comprises a coding sequence for a proteinwhose production is desired in the cells of the intestinal tract linkedto necessary regulatory sequences for expression in the cells. Foruptake by cells of the intestinal tract, the compositions areadministered orally or by enema whereby they enter the intestinal tractand contact cells which comprise GCC. The delivery vehicles associatewith the GCC by virtue of the compound of the invention and the vehicleis internalized into the cell or the active agent/genetic construct isotherwise taken up by the cell. Once internalized, the construct canprovide a therapeutic effect on the individual.

Antisense

The present invention provides compositions, kits and methods which areuseful to prevent and treat colorectal, stomach or esophageal cancercells by providing the means to specifically deliver antisense compoundsto colorectal, stomach or esophageal cancer cells and thereby stopexpression of genes in such cells in which undesirable gene expressionis taking place without negatively effecting cells in which no suchexpression occurs.

The conjugated compositions of the present invention are useful fortargeting cells that express GCC including colorectal, stomach oresophageal cancer cells. The conjugated compositions will not bind tonon-colorectal derived cells. Non-colorectal cells, lacking GCC, do nottake up the conjugated compositions. Normal colorecial cells do have GCCand will take up the compositions. The present invention providescompositions and methods of delivering antisense compositions to normaland cancerous colorectal cells and stomach or esophageal cancer cells.

The present invention provides a cell specific approach in which onlynormal and cancerous colorectal cells and primary and/or metastaticstomach or esophageal cancer cells are exposed to the active portion ofthe compound and only those cells are effected by the conjugatedcompound. The compound of the invention binds to normal and cancerouscolorectal cells and primary and/or metastatic stomach or esophagealcancer cells. Upon binding to these cells, the conjugated compound isinternalized and the delivery of the conjugated compound including theantisense portion of the molecule is effected. The presence of theconjugated compound in normal colorectal cells has no effect on suchcells because the cancer-associated gene for which the antisensemolecule that makes up the active moiety of the conjugated compound iscomplementary is not being expressed. However, in colorectal cancercells, the cancer gene for which the antisense molecule that makes upthe active moiety of the conjugated compound is complementary is beingexpressed. The presence of the conjugated compound in colorectal cancercells serves to inhibit or prevent transcription or translation of thecancer gene and thereby reduce or eliminate the transformed phenotype.

The invention can be used to combat primary and/or metastasizedcolorectal, stomach or esophageal cancer as well as to prevent theemergence of the transformed phenotype in normal colon cells. Thus theinvention can be used therapeutically as well as prophylactically.

One having ordinary skill in the art can readily identify individualssuspected of suffering from stomach or esophageal cancer. In thoseindividuals diagnosed with stomach or esophageal cancer, it is standardtherapy to suspect metastasis and aggressively attempt to eradicatemetastasized cells. The present invention provides pharmaceuticalcompositions and methods for specifically targeting and eliminatingmetastasized colorectal cancer cells and primary and/or metastaticstomach or esophageal cancer cells. Further, the present inventionprovides pharmaceutical compositions that comprise therapeutics andmethods for specifically eliminating metastasized colorectal cancercells and primary and/or metastatic stomach or esophageal cancer cells.

The present invention relies upon the use of a compound of the inventionin a conjugated composition. The compound of the invention isessentially a portion of the conjugated composition which acts as aligand to GCC and thus specifically binds to these receptors. Theconjugated composition also includes an active moiety which isassociated with the compound of the invention; the active moiety beingan antisense composition useful to inhibit or prevent transcription ortranslation of expression of genes whose expression is associated withcancer.

According to the present invention, the active moiety is an antisensecomposition. In particular, the antisense molecule that makes up theactive moiety of a conjugated compound hybridizes to DNA or RNA in acolorectal, stomach or esophageal cancer cell and inhibits and/orprevents transcription or translation of the DNA or RNA from takingplace. The antisense compositions may be a nucleic acid molecule, aderivative or an analogs thereof. The chemical nature of the antisensecomposition may be that of a nucleic acid molecule or a modified nucleicacid molecule or a non-nucleic acid molecule which possess functionalgroups that mimic a DNA or RNA molecule that is complementary to the DNAor RNA molecule whose expression is to be inhibited or otherwiseprevented. Antisense compositions inhibit or prevent transcription ortranslation of genes whose expression is linked to colorectal, stomachor esophageal cancer, i.e. cancer associated genes.

Point mutations insertions, and deletions in K-ras and H-ras have beenidentified in many tumors. Complex characteristics of the alterations ofoncogenes HER-2/ERBB-2, HER-1/ERBB-1, HRAS-1, C-MYC and anti-oncogenesp53, RB1.

Chemical carcinogenesis in a rat model demonstrated point mutations infos, an oncogene which mediates transcriptional regulation andproliferation. See: Alexander, R J, et al. Oncogene alterations in ratcolon tumors induced by N-methyl-N-nitrosourea. American Journal of theMedical Sciences. 303(1):16-24, 1992, January which is herebyincorporated herein by reference including all references cited thereinwhich are also hereby incorporated herein by reference.

Chemical carcinogenesis in a rat model demonstrated point mutations inthe oncogene abl. See: Alexander, R J, et al. Oncogene alterations inrat colon tumors induced by N-methyl-N-nitrosourea. American Journal ofthe Medical Sciences. 303(1):16-24, 1992, January.

MYC is an oncogene that plays a role in regulating transcription andproliferation. A 15-base antisense oligonucleotide to myc complementaryto the translation initiation region of exon II was incubated withcolorectal cancer cells. This antisense molecule inhibited proliferationof colorectal cancer cells in a dos-dependent fashion. Interestingly,the uptake of this oligonucleotide was low (0.7%). Also, transfer of anormal chromosome 5 to colorectal cancer cells resulted in theregulation of myc expression and loss of proliferation. These datasuggest that a tumor suppressor gene important in the regulation of mycis contained on this chromosome.

A novel protein tyrosine phosphatase, G1, has been identified.Examination of the mRNA encoding this protein in colorectal tumor cellsrevealed that it undergoes point mutations and deletions in these cellsand may play a role in proliferation characteristic of these cells.Takekawa, M. et al. Chromosomal localization of the protein tyrosinephosphatase G1 gene and characterization of the aberrant transcripts inhuman colon cancer cells. FEBS Letters. 339(3):222-8, 1994 Feb. 21,which is hereby incorporated herein by reference including allreferences cited therein which are also hereby incorporated herein byreference.

Gastrin regulates colon cancer cell growth through a cyclicAMP-dependent mechanism mediated by PKA. Antisense oligodeoxynucleotidesto the regulatory subunit of a specific class of PKA inhibited thegrowth-promoting effects of cyclic AMP in colon carcinoma cells. See:Bold, R J, et al. Experimental gene therapy of human colon cancer.Surgery. 116(2):189-95; discussion 195-6, 1994 August and Yokozaki, H.,et al. An antisense oligodeoxynucleotide that depletes R1 alpha subunitof cyclic AMP-dependent protein kinase induces growth inhibition inhuman cancer cells. Cancer Research. 53(4):868-72, 1993 Feb. 15, whichare both hereby incorporated herein by reference including allreferences cited therein which are also hereby incorporated herein byreference.

CRIPTO is an epidermal growth factor-related gene expressed in amajority of colorectal cancer tumors. Antisense phosphorothioateoligodeoxynucleotides to the 5′-end of CRIPTO mRNA significantly reducedCRIPTO expression and inhibited colorectal tumor cell growth in vitroand in vivo. Ciardiello, F. et al. Inhibition of CRIPTO expression andtumorigenicity in human colon cancer cells by antisense RNA andoligodeoxynucleotides. Oncogene. 9(1):291-8, 1994 January which are bothhereby incorporated herein by reference including all references citedtherein which are also hereby incorporated herein by reference.

Many carcinoma cells secrete transforming growth factor alpha. A 23nucleotide antisense oligonucleotide to TGF alpha mRNA inhibited bothDNA synthesis and proliferation of colorectal cancer cells. Sizeland, AM, Burgess, A W. Antisense transforming growth factor alphaoligonucleotides inhibit autocrine stimulated proliferation of a coloncarcinoma cell line. Molecular Biology of the Cell. 3(11):1235-43, 1992November which is hereby incorporated herein by reference including allreferences cited therein which are also hereby incorporated herein byreference.

Antisense compositions including oligonucleotides, derivatives andanalogs thereof, conjugation protocols, and antisense strategies forinhibition of transcription and translation are generally described in:Antisense Research and Applications, Crooke, S. and B. Lebleu, eds. CRCPress, Inc. Boca Raton Fla. 1993, Nucleic Acids in Chemistry and BiologyBlackburn, G. and M. J. Gait, eds. IRL Press at Oxford University Press,Inc. New York 1990; and Oligonucleotides and Analoguesn: A PracticalApproach Eckstein, F. ed., IRL Press at Oxford University Press, Inc.New York 1991; which are each hereby incorporated herein by referenceincluding all references cited therein which are hereby incorporatedherein by reference.

The antisense molecules of the present invention comprise a sequencecomplementary to a fragment of a colorectal cancer gene. See Ullrich etal., EMBO J., 1986, 5:2503, which is hereby incorporated herein byreference.

Antisense compositions which can make up an active moiety in conjugatedcompounds of the invention include oligonucleotides formed ofhomopyrimidines can recognize local stretches of homopurines in the DNAdouble helix and bind to them in the major groove to form a triplehelix. See: Helen, C and Toulme, J J. Specific regulation of geneexpression by antisense, sense, and antigene nucleic acids. Biochem.Biophys Acta, 1049:99-125, 1990 which is hereby incorporated herein byreference including all references cited therein which are herebyincorporated herein by reference. Formation of the triple helix wouldinterrupt the ability of the specific gene to undergo transcription byRNA polymerase. Triple helix formation using myc-specificoligonucleotides has been observed. See: Cooney, M, et al. Science241:456-459 which is hereby incorporated herein by reference includingall references cited therein which are hereby incorporated herein byreference.

Antisense oligonucleotides of DNA or RNA complementary to sequences atthe boundary between introns and exons can be employed to prevent thematuration of newly-generated nuclear RNA transcripts of specific genesinto mRNA for transcription. Antisense RNA complimentary to specificgenes can hybridize with the mRNA for tat gene and prevent itstranslation. Antisense RNA can be provided to the cell as “ready-to-use”RNA synthesized in vitro or as an antisense gene stably transfected intocells which will yield antisense RNA upon transcription. Hybridizationwith mRNA results in degradation of the hybridized molecule by RNAse Hand/or inhibition of the formation of translation complexes. Both resultin a failure to produce the product of the original gene.

Antisense sequences of DNA or RNA can be delivered to cells. Severalchemical modifications have been developed to prolong the stability andimprove the function of these molecules without interfering in theirability to recognize specific sequences. These include increasing theirresistance to degradation by DNases, including phosphotriesters,methylphosphonates, phosphorothioates, alpha-anomers, increasing theiraffinity for their target by covalent linkage to various intercalatingagents such as psoralens, and increasing uptake by cells by conjugationto various groups including polylysine. These molecules recognizespecific sequences encoded in mRNA and their hybridization preventstranslation of and increases the degradation of these messages.

Conjugated compositions of the invention provide a specific andeffective means for terminating the expression of genes which causeneoplastic transformation. GCC undergo ligand-induced endocytosis andcan deliver conjugated compounds to the cytoplasm of cells.

Compound of the inventions are conjugated directly to antisensecompositions such as nucleic acids which are active in inducing aresponse. For example, antisense oligonucleotides to MYC are conjugateddirectly to a compound of the invention. This has been performedemploying peptides that bind to the CD4 receptor. See: Cohen, J S, ed.Oligodeoxynucleotides: Antisense Inhibitors of Gene Expression. Topicsin Molecular and Structural Biology. CRC Press, Inc., Boca Raton, 1989.which is hereby incorporated herein by reference including allreferences cited therein which are hereby incorporated herein byreference. The precise backbone and its synthesis is not specified andcan be selected from well-established techniques. Synthesis wouldinvolve either chemical conjugation or direct synthesis of the chimericmolecule by solid phase synthesis employing FMOC chemistry. See:Haralambidis, J, et al. (1987) Tetrahedron Lett. 28:5199-5202, which ishereby incorporated herein by reference including all references citedtherein which are hereby incorporated herein by reference.Alternatively, the peptide-nucleic acid conjugate may be synthesizeddirectly by solid phase synthesis as a peptide-peptide nucleic acidchimera by solid phase synthesis. Nielsen, P E, et al. (1994)Sequence-specific transcription arrest by peptide nucleic acid bound tothe DNA template strand. Gene 149:139-145, which is hereby incorporatedherein by reference including all references cited therein which arehereby incorporated herein by reference.

In some embodiments, polylysine can be complexed to conjugatedcompositions of the invention in a non-covalent fashion to nucleic acidsand used to enhance delivery of these molecules to the cytoplasm ofcells. In addition, peptides and proteins can be conjugated topolylysine in a covalent fashion and this conjugate complexed withnucleic acids in a non-covalent fashion to further enhance thespecificity and efficiency of uptake of the nucleic acids into cells.Thus, the compound of the invention is conjugated chemically topolylysine by established techniques. The polylysine-compound of theinvention conjugate may be complexed with nucleic acids of choice. Thus,polylysine-orosomucoid conjugates were employed to specifically plasmidscontaining genes to be expressed to hepatoma cells expressing theorosomucoid receptor. This approach can be used to delivery whole genes,or oligonucleotides. Thus, it has the potential to terminate theexpression of an undesired gene (eg. MYC, ras) or replace the functionof a lost or deleted gene (eg. hMSH2, hMLH1, hPMS1, and hPMS2).

According to a preferred embodiment, Myc serves as a gene whoseexpression is inhibited by an antisense molecule within a conjugatedcomposition. Compound of the inventions are used to deliver a 15-basedantisense oligonucleotide to myc complementary to the translationinitiation region of exon II. The 15-base antisense oligonucleotide toMYC is synthesized as reported in Collins, J F, Herman, P, Schuch, C,Bagby G C, Jr. Journal of Clinical Investigation. 89(5):1523-7, 1992May. In some embodiments, the conjugated composition is conjugated topolylysine as reported previously. Wu, G Y, and Wu, C H. (1988) Evidencefor ed gene delivery to Hep G2 hepatoma cells in vitro. Biochem.27:887-892 which is incorporated herein by reference.

Conjugated compositions may be synthesized as a chimeric moleculedirectly by solid phase synthesis. pmolar to nanomolar concentrationsfor this conjugate suppress MYC synthesis in colorectal cancer cells invitro.

Antisense molecules are preferably hybridize to, i.e. are complementaryto, a nucleotide sequence that is 5-50 nucleotides in length, morepreferably 5-25 nucleotides and in some embodiments 10-15 nucleotides.

In addition, mismatches within the sequences identified above, whichachieve the methods of the invention, such that the mismatched sequencesare substantially complementary to the cancer gene sequences are alsoconsidered within the scope of the disclosure. Mismatches which permitsubstantial complementarity to the cancer gene sequences will be knownto those of skill in the art once armed with the present disclosure. Theoligos may also be unmodified or modified.

Therapeutic compositions and methods may be used to combat colorectal,stomach or esophageal cancer in cases where the cancer is localizedand/or metastasized. Individuals are administered a therapeuticallyeffective amount of conjugated compound. A therapeutically effectiveamount is an amount which is effective to cause a cytotoxic orcytostatic effect on cancer cells without causing lethal side effects onthe individual. An individual who has been administered atherapeutically effective amount of a conjugated composition has aincreased chance of eliminating colorectal, stomach or esophageal canceras compared to the risk had the individual not received thetherapeutically effective amount.

To treat localized colorectal, stomach or esophageal cancer, atherapeutically effective amount of a conjugated compound isadministered such that it will come into contact with the localizedtumor. Thus, the conjugated compound may be administered orally orintratumorally. Oral and rectal formulation are taught in Remington'sPharmaceutical Sciences, 18th Edition, 1990, Mack Publishing Co., EastonPa. which is incorporated herein by reference.

The pharmaceutical compositions according to the present invention maybe administered as either a single dose or in multiple doses. Thepharmaceutical compositions of the present invention may be administeredeither as individual therapeutic agents or in combination with othertherapeutic agents. The treatments of the present invention may becombined with conventional therapies, which may be administeredsequentially or simultaneously.

The present invention is directed to a method of delivering antisensecompounds to normal and cancerous colorectal cells and to stomach oresophageal cancer cells and inhibiting expression of cancer genes inmammals. The methods comprise administering to a mammal an effectiveamount of a conjugated composition which comprises a compound of theinvention conjugated to an antisense oligonucleotide having a sequencewhich is complementary to a region of DNA or mRNA of a cancer gene.

The conjugated compounds may be administering to mammals in a mixturewith a pharmaceutically-acceptable carrier, selected with regard to theintended route of administration and the standard pharmaceuticalpractice. Dosages will be set with regard to weight, and clinicalcondition of the patient. The conjugated compositions of the presentinvention will be administered for a time sufficient for the mammals tobe free of undifferentiated cells and/or cells having an abnormalphenotype. In therapeutic methods treatment extends for a timesufficient to inhibit transformed cells from proliferating andconjugated compositions may be administered in conjunction with otherchemotherapeutic agents to manage and combat the patient's cancer.

The conjugated compounds of the invention may be employed in the methodof the invention singly or in combination with other compounds. Theamount to be administered will also depend on such factors as the age,weight, and clinical condition of the patient. See Gennaro, Alfonso,ed., Remington's Pharmaceutical Sciences, 18th Edition, 1990, MackPublishing Co., Easton, Pa.

Methods and Compositions for Making and Using Targeted Gene Therapy,Antisense and Drug Compositions.

Liposomes may be prepared by well known means wherein Compound of theinventions may be exposed on the outside of the liposomes. Suchliposomes may contain any of the active agents descried herein in othersections of this specification. Alternatively, genetic constructs may beprovided within the liposomes to deliver gene therapeutics tonon-malignant and malignant intestinal cells or extraintestinal cellssuch as stomach or espophageal cancer cells, which express GCC. Deliveryto non-malignant intestinal cells may be useful to provide such cellswith functional genes to express proteins for which the individual islacking an functional gene, to supplement the individuals gene or toprovide a therapeutic protein. For example, in patients suffering cysticfibrosis, the incorporation of a DNase gene in the intestinal cells mayprovide therapeutic benefit to the individual. Similarly, in patientswith intestinal disorders such as inflammatory bowel disease or Crohn'sdisease, genes which can down regulate immune response or protect cellsagainst the self-directed immune response may be useful.

Methods of Preventing and Treating Diahhrea

Accordingly, the present invention relates to a method of negativelyregulating guanylyl cyclase C (GC-C) enzyme activity by activating aninhibitory pathway. According to the present invention, GC-C enzymeactivity is inhibited by contacting cells that have active GC-C enzymewith an effective amount of a compound of the invention or apharmaceutically acceptable salt thereof. GC-C compound of theinventions are useful to prevent or treat OC—C-mediated diarrhea. Thepresent invention relates to a method of preventing an individual fromsuffering from GC-C-mediated diarrhea, such as infectious diarrhea,comprising the step of administering to such an individual, an amount ofa compound of the invention, or a pharmaceutically acceptable saltthereof to inhibit GC-C activity. The present invention relates to amethod of treating an individual suffering from GC-C-mediated diarrhea,such as infectious diarrhea, comprising the step of administering tosuch an individual, an amount of a compound of the invention, or apharmaceutically acceptable salt thereof to inhibit GC-C activity.

According to some aspects of the invention, methods are provided toprevent diarrhea in humans induced by ST producing organisms indeveloping countries; to prevent growth retardation in children indeveloping countries in which ST-producing organisms are endemic; toprevent travelers' diarrhea; and to prevent scours and other diarrhealdiseases in animals; as well as to treat secretory diarrhea of unknownetiology; and to treat irritable bowel syndrome and other forms ofdiarrhea including, but not limited to, diarrhea associated withinflammatory bowel disease, sprue, etc.

The method of the present invention comprises inhibiting GC-C activityby contacting cells that have active GC-C with an effective amount of acompound of the invention, or a pharmaceutically acceptable saltthereof. Antagonist compounds according to the invention have beendiscovered to negatively regulate the activity of GC-C. Exposure ofcells in vitro a compound of the invention has resulted in theinhibition of GC-C activity. Inhibition of GC-C activity in cellsimpedes the pathway associated with GC-C-mediated diarrhea.

The method that is the present invention is useful in the prevention andtreatment of conditions associated with active GC-C; specificallyGC-C-mediated diarrhea such as infectious diarrhea. Accordingly, thepresent invention relates to a method of preventing GC-C-mediateddiarrhea in or treating a individual suffering from GC-C-mediateddiarrhea. The present invention relates to a method of preventinginfectious in an a individual susceptible to infectious diarrhea. Thepresent invention relates to a method of treating an individualsuffering from infectious diarrhea. The individual is preferably amammal, more preferably a human. In some embodiments, the individual maybe a canine, feline, bovine, ovine or equine species.

The identification of an individual suspected of suffering fromGC-C-mediated diarrhea such as infectious diarrhea is routine and can beperformed by those having ordinary skill in the art. Infectiousdiarrhea, also referred to as travelers diarrhea, can be identifiedroutinely. In addition to being useful to treating individuals suspectedof suffering from GC-C-mediated diarrhea, the present invention isuseful prophylactically to prevent the incidence of GC-C-mediateddiarrhea in individuals at risk of contracting the condition. Forexample, travelers and those living in situations for which they areunaccustomed may be at risk. Those having ordinary skill in the art canreadily identify conditions which place an individual at risk ofcontracting GC-C-mediated infectious diarrhea.

In some preferred embodiments, the method that is the invention is amethod of treating an individual suspected of suffering from GC-Cmediated diarrhea comprising the step of administering to saidindividual, an effective amount of a compound of the invention compound.In addition, pharmaceutically acceptable salts of these compounds may beused in the method that is the present invention.

Pharmaceutically acceptable salts of these compounds may be used inpracticing the method that is the present invention. Pharmaceuticalcompositions containing the compounds or salts may also be used inpracticing the method that is the present invention. Pharmaceuticallyacceptable salts useful in the method of that is the invention includesodium, potassium, calcium, zinc, lithium, magnesium, aluminum,diethanolamine, tromethamine, ethylenediamine, meglumine, hydrochloric,hydrobromic or acetic acid.

The present invention relates to a method of using antagonist compoundsof the invention to inhibit the activity of GC-C in cells. The range ofamounts of compound of the invention that a cell can be exposed to thatis effective for inhibiting GC-C activity can be determined by onehaving ordinary skill in the art.

By inhibiting GC-C activity, the method that is the present invention isuseful in the treatment of GC-C-mediated diarrhea. Effective amounts ofcompound of the inventions used in the method that is the presentinvention can be formulated as pharmaceutical preparations andadministered to individuals who are suspected of suffering from or beingsusceptible to GC-C mediated diarrhea, in order to counter thebiochemical pathway which leads to the condition at the cellular level.

Treatment of GC-C-mediated diarrhea can be performed by administrationof effective amounts of a pharmaceutical preparation of compound of theinvention to inhibit GC-C. Compounds can be formulated for human andanimal prophylactic and therapeutic applications by those havingordinary skill in the art. The dosage range of a compound to beadministered to mammals, particularly humans, to be effective in thetreatment or prevention of GC-C mediated diarrhea can be determined bythose having ordinary skill in the art.

Pharmaceutical preparations incorporating compounds used in the methodthat is the invention can be used to block GC-C activity related to thebiochemical pathway which results in diarrhea by administration ofeffective amounts of pharmaceutical preparation that comprise compoundsdisclosed herein. The compounds used in the method that is the inventioncan be formulated for human and animal prophylactic and therapeuticapplications by those having ordinary skill in the art. The range ofamounts of a compound to be administered to mammals, particularlyhumans, to be effective in treating or preventing GC-C-mediated diarrheacan be determined by those having ordinary skill in the art.

The mode of administration of compounds and pharmaceutical compositionsaccording to the method that is the invention includes any means thatproduces contact of the active ingredient with the agent's site ofaction in the body of a mammal, that is in the cells of the colon. Thesemodes of administration include but not limited to oral, topical,hypodermal, intravenous, intraanally, intramuscular and intraparenteralmethods of administration. The preferred route of administration isorally.

In practicing the method that is the invention, the compounds may beadministered singly or in combination with other compounds useful fortreating or preventing diarrhea. In the method that is the invention,the compounds are preferably administered with a pharmaceuticallyacceptable carrier selected on the basis of the selected route ofadministration and standard pharmaceutical practice.

The method may include administration of compounds to mammals,preferably humans, in therapeutically effective amounts which areeffective to inhibit GC-C. The dosage administered in any particularinstance will depend upon factors such as the pharmacodynamiccharacteristics of the compound of the invention, its mode and route ofadministration; age, health, and weight of the recipient; nature andextent of symptoms; kind of concurrent treatment, frequency oftreatment, and the effect desired.

It is contemplated that the daily dosage of a compound used in themethod that is the invention will be in the range of from about 1micrograms to about 10 grams per day. In some preferred embodiments, thedaily dosage compound will be in the range of from about 10 mg to about1 gram per day. In some preferred embodiments, the daily dosage compoundwill be in the range of from about 100 mg to about 500 mg per day. It iscontemplated that the daily dosage of a compound used in the method thatis the invention will be in the range of from about 1 μg to about 100 mgper kg of body weight, in some embodiments, from about 1 μg to about 40mg per kg body weight; in some embodiments from about 10 μg to about 20mg per kg per day, and in some embodiments 10 μg to about 1 mg per kgper day.

Pharmaceutical compositions may be administered in a single dosage,divided dosages or in sustained release. In some preferred embodiments,the compound will be administered in multiple doses per day. In somepreferred embodiments, the compound will be administered in 3-4 dosesper day.

The method of administering compounds include administration as apharmaceutical composition orally in solid dosage forms, such ascapsules, tablets, and powders, or in liquid dosage forms, such aselixirs, syrups, and suspensions. The compounds may also be administeredparenterally in sterile liquid dosage forms or topically in a carrier.The compounds may be formulated into dosage forms according to standardpractices in the field of pharmaceutical preparations. See Remington'sPharmaceutical Sciences, A. Osol, Mack Publishing Company, Easton, Pa.

Compounds may be mixed with powdered carriers, such as lactose, sucrose,mannitol, starch, cellulose derivatives, magnesium stearate, and stearicacid for insertion into gelatin capsules, or for forming into tablets.Both tablets and capsules may be manufactured as sustained releaseproducts for continuous release of medication over a period of hours.Compressed tablets can be sugar or film coated to mask any unpleasanttaste and protect the tablet from the atmosphere or enteric coated forselective disintegration in the gastrointestinal tract. In somepreferred embodiments, compounds are delivered orally and are coatedwith an enteric coating which makes the compounds available upon passingthrough the stomach and entering the intestinal tract, preferably uponentering the large intestine. U.S. Pat. No. 4,079,125, which isincorporated herein by reference, teaches enteric coating which may beused to prepare enteric coated compound of the inventions useful in themethods of the invention.

Liquid dosage forms for oral administration may contain coloring andflavoring to increase patient acceptance, in addition to apharmaceutically acceptable diluent such as water, buffer or salinesolution.

For parenteral administration, a compound may be mixed with a suitablecarrier or diluent such as water, a oil, saline solution, aqueousdextrose (glucose), and related sugar solutions, and glycols such aspropylene glycol or polyethylene glycols. Solutions for parenteraladministration contain preferably a water soluble salt of the compound.Stabilizing agents, antioxidizing agents and preservatives may also beadded. Suitable antioxidizing agents include sodium bisulfite, sodiumsulfite, and ascorbic acid, citric acid and its salts, and sodium EDTA.Suitable preservatives include benzalkonium chloride, methyl- orpropyl-paraben, and chlorbutanol.

According to some aspects of the invention, the prevention of diarrheaand the dehydration that accompanies it in children prevents growthretardation, a significant problem among in children in developingcountries in which ST-producing organisms are endemic. Travelers canprevent travelers' diarrhea be prophylactical administration of compoundof the inventions prior to and during travel to locations whereST-producing organisms are endemic. The administration of compound ofthe inventions can be used to prevent scours and other diarrhealdiseases in animals identified as being susceptible to the same.Individuals suffering from secretory diarrhea of unknown etiology may betreated by administration of compound of the inventions. Aspects of thepresent invention include treatment of individuals identified as havingirritable bowel syndrome or other forms of diarrhea be administering tothem therapeutic amounts of compound of the invention.

Aspects of the present invention include methods of imaging proximalrenal tubules and treating renal diseases and disorders involving thesame such as some renal cancer, methods of imaging exocrine duct cellsof the pancreas and treating pancreatic diseases and disorders involvingthe same such as some pancreatic cancer, methods of imagingsubmandibular glands and treating diseases and disorders involving thesame, and the methods of imaging bile duct and treating diseases anddisorders involving the same such as some gall bladder cancers. In eachcase, the teachings above may be generally applied and adapted from thethose discussed above.

EXAMPLES Example 1

Compound of the inventions of GCC that binds to the receptor with highaffinity but does not induce the production of cyclic GMP has beendesigned. The peptides block the ability of ST, the most potent agonistof GCC known, to bind to GCC and induce accumulation of cyclic GMP.Thus, the peptides fulfill the criteria for a compound of the inventionof GCC. The structure of these compound of the inventions identify acore peptide mediating binding to GCC and the agonist switch whichinduces the production of cyclic GMP.

The molecules were derived from ST. Comparison of the structure of thecompound of the invention with the known agonists reveal the coredeterminants required for receptor occupancy and agonist activation. Allknown biologically-active GCC agonists possess intrachain disulfidesbetween cysteines at positions 6 and 14 and positions 9 and 17. Inaddition, some agonists, such as the STs, have an intrachain disulfidebetween cysteines at position 5 and 10. The compound of the invention,which binds to GCC but does not activate cyclic GMP production, retainsthe intrachain disulfides at positions 5 and 10 and positions 6 and 14,but not the disulfide at positions 9 and 17. These data demonstrate thatthe core structure consisting of residues 5-17 and possessing disulfidesat positions 5 and 10 and positions 6 and 14 encompasses thedeterminants to bind to GCC, but that the structure formed by andencompassing the positions 9 and 17 intrachain disulfide contains theagonist switch that activates GCC to produce cyclic GMP.

The identification of this structure provides a platform for developingagonists and compound of the inventions for GCC which exhibit greaterpotency, efficacy and specificity than existing ligands. In addition,this structure provides a platform for developing non-peptide agonistsand compound of the inventions, conferring greater stability tofacilitate oral, intravenous, subcutaneous or other delivery in theabsence of degradation by proteases.

The structures of the compound of the inventions are set forth in Table2 together with the structure for the E. coli heat stable entertoxinSTa. The numerical positions refer to those of the Sta toxin. Position 56 9 10 14 17 E. coli STa N T F Y C C E L C C N P A C A G C Y ST(4-14)Ala9,17 C C E L A C N P A C A G A ST(4-14) NMeLeu9 Y C C E L L* C N P AC ST(4-14) NMeAla9 Y C C E L A* C N P A C A G A ST(4-14) Phe9Cys(Mob)5,10 Mono Y C† C E L F C† N P A C A G A ST(4-14) Phe9 Y C C E LF C N P A C ST(4-14) NMeVal9 Y C C E L V* C N P A C ST(5-14) Ala9Tyr15 CC E L A C N P A C Y ST(4-14) Thr9Tyr15 C C E L T C N P A C Y ST(4-14)Thr9 Aib13e Y C C E L T C N P α C ST(4-14) Thr9 Aib12 Y C C E L T C N αA C ST(4-14) Leu9Tyr15 Y C C E L L C N P A C Y ST(4-14) Ala9 NMeAla13 YC C E L A C N P A* C ST(5-14) Ala9 C C E L A C N P A C ST(6-14) Mpr5Ala9 Carba5,10 γδ C E L A δ N P A C* indicates amino acid is N methylated† indicates amino acid is blocked with Mobα indicates Aibγ indicates Mprδ indicates Carba linkage

Example 2

T84 human colorectal adenocarcinoma cells were grown to confluence inculture and harvested using a trypsin/EDTA solution. These cells werediluted 10-fold (v/v) with Dulbecco's minimal essential medium/F12, plus10% fetal bovine serum (DMEM/F12/FBS) and plated at a density of 20,000cells/well into a Falcon 96-well plate. The cells in this 96-well platewere grown to near confluence in the DMEM/F12/FBS, then washed oncesequentially with 200 microliters each of Dulbecco's phosphate bufferedsaline (No Ca or Mg) and Opti-mem serum-free media. The Opti-mem mediawas removed and replaced with 90 microliters of Opti-mem mediacontaining 1.1 mM IBMX (iso). This solution was incubated with the cellsfor 15 minutes at 37 degrees C. and then 10 microliters of an ST analog(10× desired final concentration) was added. This solution was mixed andincubated at 37 degrees Celsius for 15 minutes before the media wasaspirated off and replaced with 200 microliters of lysis reagent 1 (0.5%dodecyltrimethylammonium bromide, aqueous) from the Amersham BiosciencescGMP Enzyme Immunoassay Biotrak System and incubated at 37 degreesCelsius for 15 minutes. cGMP levels were quantified against a cGMPstandard curve using the Amersham Biosciences cGMP competitive EnzymeImmunoassay Biotrak System. This System detects cGMP in the followingmanner: a Donkey anti-rabbit IgG is adhered to the immunoassay plate andincubated with Rabbit anti-cGMP antibody. The wells are thenco-incubated with cell culture lysate and a standard amount ofcGMP-horseradish peroxidase conjugate for two hours at 4 degrees Celsiusand washed prior to addition of the substrate3,3′,5,5′-tetramethylbenzidine/hydrogen peroxide which generates a bluecolor. The addition of 100 microliters of 1 M sulfuric acid (aq)stabilizes the color and it is then read at 450 nm. The data is shown inFIG. 5.

1. A compound a structure according to formula (I)R1-R2-R3-R4-R5-R6-R7  (I) wherein: R1 is combination of 1-50 naturalamino acids, 1-50 blocked amino acids, 1-50 synthetic amino acids, 1-50derivatized amino acids; 1-50 amino acid mimetics, a 1-50 alkyls, one ormore 1-50 substituted alkyls, one or more 1-50 aryls, one or more 1-50substituted aryls, one or more 1-50 alkylaryls, one or more 1-50substituted alkylaryls or combinations thereof; R2 is a natural aminoacid, blocked amino acid, synthetic amino acid, derivatized amino acid;an amino acid mimetic; R3 is a natural amino acid, blocked amino acid,synthetic amino acid, derivatized amino acid or amino acid mimetic thatcrosslinks with R8; R4 is one or two natural amino acids, one or twoblocked amino acids, one or two synthetic amino acids, one or twoderivatized amino acids, one or two amino acid mimetics, or a dipeptidemimetic or combinations thereof, wherein R4 forms a 3₁₀-helix turn grouplinking R3 to R5; R5 is a natural amino acid, blocked amino acid,synthetic amino acid, derivatized amino acid or amino acid mimetic thatdoes not crosslink with R9 R6 is a natural amino acid, blocked aminoacid, synthetic amino acid, derivatized amino acid, or amino acidmimetic; R7 is one, two or three natural amino acids, one, two or threeblocked amino acids, one, two or three amino acid mimetics, or atripeptide mimetic or combinations thereof, wherein R7 forms a beta turngroup linking R6 to R8 R8 is a natural amino acid, blocked amino acid,synthetic amino acid, derivatized amino acid or amino acid mimetic thatcrosslinks with R3; R9 is a combination of 1-50 natural amino acids,1-50 blocked amino acids, 1-50 synthetic amino acids, 1-50 derivatizedamino acids; 1-50 amino acid mimetics, a 1-50 alkyls, one or more 1-50substituted alkyls, one or more 1-50 aryls, one or more 1-50 substitutedaryls, one or more 1-50 alkylaryls, one or more 1-50 substitutedalkylaryls or combinations thereof that does not crosslink to R5;wherein the structure of the compounds is not:C-hC-E-L-A-C-N-P-A-X; hCC-E-L-A-X-N-P-A-C;C-C-E-L-A-C-N-P-A-C;C-C-E-L-A-C-N-P-A-C-T-G-A;Y-Ca-C-E-L-F-Ca-N-P-A-C;Y-C-C-E-L-mA-C-N-P-A-C;Y-C-C-E-mL-A-C-N-P-A-C;C-C-E-L-Cm-C-N-P-A-C-A-G-Cm;C-C-E-L-Ca-C-N-P-A-C-A-G-Ca; orX-Ca-E-L-A-hC-N-P-A-Ca; wherein hC is Homocysteine; X is Alanine linkedto hC via a covalent CH2-S bond between an Ala side chain and an hC sidechain; Cm is Cysteine(S-methoxybenzyl); and Ca isCysteine(S-acetamidomethyl).
 2. The compound of claim 1 wherein saidcompound is a conjugated compound having a compound of formula (I)conjugated to an active moiety selected from the group consisting of: aradionuclide, an enzyme, a fluorescent label, a metal chelating group, achemiluminescent label, a bioluminescent label, a chemotherapeutic, atoxin, an inactive prodrug, a radiosensitizing agent, a photodynamicagent, a nucleic acid molecule or combinations thereof.
 3. The compoundof claim 1 wherein said compound is a conjugated compound having acompound of formula (I) conjugated to an active moiety selected from thegroup consisting of: ⁴³K, ⁵²Fe, ⁵⁷Co, ⁶⁷Cu, ⁶⁷Ga, ⁶⁸Ga, ⁷⁷Br, ⁸¹Rb/⁸MKr,⁸⁷MSr, ⁹⁹MTc, ¹¹¹In ¹¹³MIn, ¹²³I, ¹²⁷Cs, ¹²⁹Cs, ¹³¹I, ¹³²I, ¹⁹⁷Hg,²⁰³Pb, ²⁰⁶206Bi, ⁴⁷Sc, ⁹⁰Y, ¹⁰⁹Pd, ¹²³I, ¹²⁵I, ¹³¹I, ¹⁸⁶Re, ¹⁸⁸Re,¹⁹⁹Au, ²¹¹At, ²¹²Pb and ²¹²Bi, ³²P, ³³P, ⁷¹Ge, ⁷⁷As, ¹⁰³Pb, ¹⁰⁵Rh,¹¹¹Ag, ¹¹⁹Sb, ¹²¹Sn, ¹³¹Cs, ¹⁴³Pr, 161Tb, ¹⁷⁷Lu, ¹⁹¹Os, ¹⁹³ MPt, ¹⁹⁷Hg;a beta negative emitter, an auger emitters; malate dehydrogenase,staphylococcal nuclease, delta-5-steroid isomerase, yeast alcoholdehydrogenase, alpha-glycerophosphate dehydrogenase, triose phosphateisomerase, horseradish peroxidase, alkaline phosphatase, asparaginase,glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase,glucose-6-phosphate dehydrogenase, glucoamylase andacetylcholinesterase, fluorescein isothiocyanate, rhodamine,phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde andfluorescamine; fluorescence-emitting metals;diethylenetriaminepentaacetic acid (DTPA), ethylenediamine-tetraaceticacid (EDTA), luminol, isoluminol, theromatic acridinium ester,imidazole, acridinium salt and oxalate ester; luciferin, luciferaseaequorin, methotrexate (amethopterin), doxorubicin (adrimycin),daunorubicin, cytosinarabinoside, etoposide, 5-4 fluorouracil,melphalan, chlorambucil, cyclophosphamide, cis-platinum, vindesine,mitomycin, bleomycin, purothionin, macromomycin. 1,4-benzoquinonederivatives, trenimon, ricin, ricin A chain, Pseudomonas exotoxin (PE),diphtheria toxin (DT), Clostridium perfringens phospholipase C (PLC),bovine pancreatic ribonuclease (BPR), pokeweed antiviral protein (PAP),abrin, abrin A chain (abrin toxin), cobra venom factor (CVF), gelonin(GEL), saporin (SAP), modeccin, viscumin and volkensin;etoposidephosphate, nitroimidazoles, metronidazole, misonidazole; ⁴³K,⁵²Fe, ⁵⁷Co, ⁶⁷Cu, ⁶⁷Ga, ⁶⁸Ga ⁷⁷Br, ⁸Rb/⁸¹MKr, ⁸⁷MSr, ⁹⁹MTc, ¹¹¹In,¹¹³MIn, ¹²³I, ¹²⁷Cs, ¹²⁹Cs, ¹³¹I, ¹³²I, ¹⁹⁷Hg, ²⁰³Pb, ²⁰⁶206Bi, ⁴⁷Sc,⁹⁰Y, ¹⁰⁹Pd, ¹²³I, ¹²⁵I, ¹³¹I, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁹⁹Au, ²¹¹At, ²¹²Pb and²¹²Bi, ³²P, ³³P, ⁷¹Ge, ⁷⁷As, ¹⁰³Pb, ¹⁰⁵Rh, ¹¹¹Ag, ¹¹⁹Sb, ¹²¹Sn, ¹³¹Cs,¹⁴³Pr, ¹⁶¹Tb, ¹⁷⁷Lu, ¹⁹¹Os, ¹⁹³MPt, ¹⁹⁷Hg, ⁴⁷Sc and ²⁰⁶206Bi,fluorophore hematoporphyrin derivative (HPD), porfimer sodium(Photofrin.RTM.), BPD verteporfin and tetramethylrotamine.
 4. Acompounds of any one of claims 1-3 wherein R1 is smaller than 1000daltons. R2 is a Cysteine side chain analog [—(CR2)n-S, or —(CR2)n-H;where n is C1-C10, R is any atom and H is a reactive group], or aryl,alkyl and arylalkyl groups that may or may not crosslink to Cys10; R3 isa Cysteine side chain analog [—(CR2)n-S, or —(CR2)n-H; where n isC1-C10, R is any atom and H is a reactive group], an alpha, beta orgamma amino acid capable of forming a crosslink with Cys14 via a sidechain functional group, an amino acid mimetic which constrain thedihedral angles or the side chain to favorable configurations; R4 is asingle amino acid (all types), dipeptide, dipeptide mimetic and chainlinks, particularly structures with 4-8 atoms bridging the gap betweenR3 and R5, most preferably structures with 6 atoms bridging the gapbetween R3 and R5; R5 is a single amino acid (all types), preferablyhydrophobic or neutral ones, and their N-alkyl, N-hydroxyl, or N-arylanalogs, an amino acid mimetic which constrain the dihedral angles orthe side chain to favorable configurations; R6 is a single amino acid(all types), or an N-alkyl, N-hydroxyl, or N-aryl analog of one, anamino acid mimetic which constrain the dihedral angles or the side chainto favorable configurations R7 is a dipeptide mimetic, such asbicycle-L-seryl-proline, Btd, APM, ACTB, or ACDN or have the formulaX-Y-Z wherein X is a single amino acid (all types), preferably Asn, Asp,Ala, His, GlnGly, beta-Alanine), or a short one (Ala, Ser, Thr, Asp),and their N-alkyl, N-hydroxyl, or N-aryl analogs or a synthetic aminoacids, Y is a single amino acids (all types), preferably Pro, Ile, Ala,Val (both D and L) and their N-alkyl, N-hydroxyl, or N-aryl analogs,Synthetic amino acid, Amino acid mimetic which constrain the dihedralangles or the side chain to favorable configurations, Z is a singleamino acid (all types), preferably with no sidechain or a short sidechain and their N-alkyl, N-hydroxyl, or N-aryl analogs, R8 is a Cysteineside chain analog [—(CR2)n-S, or —(CR2)n-H; where n is C1-C10, R is anyatom and H is a reactive group], an alpha, beta or gamma amino acidscapable of forming a crosslink with Cys 14 via a side chain functionalgroup, an amino acid mimetic which constrain the dihedral angles or theside chain to favorable configurations and R9 is preferably smaller than1000 daltons.
 5. A compounds of any one of claims 1-3 wherein R1 is Y,NTFY, SSSNY, SSDWDY, ID, PN, or QE; R2 is a 2-carboxy-3-bromo-pyridine,2-bromo-benzoic acid, 2-bromo-cyclohexanoic acid, a blocked C, crosslinked to R6 by a Carba link, any non-crosslinking amino acid ormimetic; R3 is 2-carboxy-3-thiopiperidine, or C crosslinked to C at R8;R4 is a sarcosine, ornithine and lysine or having the formula J-Kwherein J is Gin, Gly, Ser, Ala, Pro, Asp, Asn,L-octahydroindole-2-carboxyilic acid, and their N-alkyl, N-hydroxyl, orN-aryl analogs and K is Ile, Norleucine, Met, Val, Phe, Trp, Tyr, Gin,Gly, Ala, Pro and L-1,2,3,4-tetrahydroisoquinoline-3-carboxyilic acid,and their N-alkyl, N-hydroxyl, or N-aryl analogs; R5 is Ala, N(Me)-Ala,Thr, Leu, N(Me)-Leu, Phe blocked Cysteine, Serine or Threonine, orL-1,2,3,4-tetrahydroisoquinoline-3-carboxyilic acid; R6 is C crosslinked to R2, blocked C such as Cmob, cross linked to R2 by a Carba linkany non-crosslinking amino acid or mimetic. R7 is a X-Y-Z wherein X is aIsoasparagine or beta alanine. Y is Pro, Ile, Ala, Val (both D and L)and their N-alkyl, N-hydroxyl, or N-aryl analogs, homoPro, Nipecoticacid, isonipecotic acid, Oic, Tic, Aib, aminobenzoate,carboxypiperidine, azetidine carboxylate and aminocyclopentenecarboxylic acid or L-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid; Zis a Gly, beta-Alanine, Ala, Ser, Thr, Asp, and their N-alkyl,N-hydroxyl, or N-aryl analogs; R8 is a 2-carboxy-3-thiopiperidine, Ccrosslinked to C at R3 or any other moiety crosslinked to R3; and R9 is0-20 amino acids.
 6. A compounds of any one of claims 1-3 wherein R1 isY, NTFY, SSSNY, SSDWDY, ID, PN, or QE; R2 is C cross linked to R6 orCmob; R3 is a C crosslinked to C at R8; R4 is E-L, or E-NmethylLeu; R5is A L, T, F, V, I, Y, NmethylLeu, NmethylAlam, and NmethylVal; R6 ispreferably C cross linked to R2, or Cmob; R7 is N-P-A; R8 is Ccrosslinked to C at R3; R9 is preferably AGA or Y.
 7. A compositioncomprising liposomes that comprise a compound of claim 1 and an activemoiety selected from the group consisting of: a radionuclide, an enzyme,a fluorescent label, a metal chelating group, a chemiluminescent label,a bioluminescent label, a chemotherapeutic, a toxin, an inactiveprodrug, a radiosensitizing agent, a photodynamic agent, a nucleic acidmolecule or combinations thereof.
 8. A method of diagnosing cancercharacterized by expression of GCC in an individual comprising the stepof detecting GCC in an extraintestinal sample form said individual bycontacting said sample or portions thereof with a compound of claim 1 or2 and detecting the presence of said compound bound to said sample.
 9. Amethod of imaging cancer characterized by expression of GCC in anindividual comprising the step of administering to said individual acompound of claim 1 conjugated to a detectable label and detectingaccumulation of said compound at a site on said individual's body.
 10. Amethod of treating cancer characterized by expression of GCC in anindividual comprising the step of administering to said individual acompound of claim 1 conjugated to an active agent which kills orinhibits replication of cells to which it binds.
 11. A method of claim8-10 wherein cancer characterized by expression of GCC is metastaticcolorectal cancer, primary esophageal cancer, metastatic esophagealcancer, primary stomach cancer or metastatic stomach cancer.
 12. Amethod of treating an individual who has enterotoxin mediated diarrheaor who is at risk of contracting enterotoxin mediated diarrheacomprising the step of administering to said individual a compound witha structure according to formula (I)R1-R2-R3-R4-R5-R6-R7  (I) wherein: R1 is combination of 1-50 naturalamino acids, 1-50 blocked amino acids, 1-50 synthetic amino acids, 1-50derivatized amino acids; 1-50 amino acid mimetics, a 1-50 alkyls, one ormore 1-50 substituted alkyls, one or more 1-50 aryls, one or more 1-50substituted aryls, one or more 1-50 alkylaryls, one or more 1-50substituted alkylaryls or combinations thereof; R2 is a natural aminoacid, blocked amino acid, synthetic amino acid, derivatized amino acid;an amino acid mimetic; R3 is a natural amino acid, blocked amino acid,synthetic amino acid, derivatized amino acid or amino acid mimetic thatcrosslinks with R8; R4 is one or two natural amino acids, one or twoblocked amino acids, one or two synthetic amino acids, one or twoderivatized amino acids, one or two amino acid mimetics, or a dipeptidemimetic or combinations thereof, wherein R4 forms a 3 ₁₀-helix turngroup linking R3 to R5; R5 is a natural amino acid, blocked amino acid,synthetic amino acid, derivatized amino acid or amino acid mimetic thatdoes not crosslink with R9 R6 is a natural amino acid, blocked aminoacid, synthetic amino acid, derivatized amino acid, or amino acidmimetic; R7 is one, two or three natural amino acids, one, two or threeblocked amino acids, one, two or three amino acid mimetics, or atripeptide mimetic or combinations thereof, wherein R7 forms a beta turngroup linking R6 to R8 R8 is a natural amino acid, blocked amino acid,synthetic amino acid, derivatized amino acid or amino acid mimetic thatcrosslinks with R3; R9 is a combination of 1-50 natural amino acids,1-50 blocked amino acids, 1-50 synthetic amino acids, 1-50 derivatizedamino acids; 1-50 amino acid mimetics, a 1-50 alkyls, one or more 1-50substituted alkyls, one or more 1-50 aryls, one or more 1-50 substitutedaryls, one or more 1-50 alkylaryls, one or more 1-50 substitutedalkylaryls or combinations thereof that does not crosslink to R5.
 13. Amethod of claim 12 wherein R1 is smaller than 1000 daltons. R2 is aCysteine side chain analog [—(CR2)n-S, or —(CR2)n-H; where n is C1-C10,R is any atom and H is a reactive group], or aryl, alkyl and arylalkylgroups that may or may not crosslink to Cys10; R3 is a Cysteine sidechain analog [—(CR2)n-S, or —(CR2)n-H; where n is C1-C10, R is any atomand H is a reactive group], an alpha, beta or gamma amino acid capableof forming a crosslink with Cys14 via a side chain functional group, anamino acid mimetic which constrain the dihedral angles or the side chainto favorable configurations; R4 is a single amino acid (all types),dipeptide, dipeptide mimetic and chain links, particularly structureswith 4-8 atoms bridging the gap between R3 and R5, most preferablystructures with 6 atoms bridging the gap between R3 and R5; R5 is asingle amino acid (all types), preferably hydrophobic or neutral ones,and their N-alkyl, N-hydroxyl, or N-aryl analogs, an amino acid mimeticwhich constrain the dihedral angles or the side chain to favorableconfigurations; R6 is a single amino acid (all types), or an N-alkyl,N-hydroxyl, or N-aryl analog of one, an amino acid mimetic whichconstrain the dihedral angles or the side chain to favorableconfigurations R7 is a dipeptide mimetic, such asbicycle-L-seryl-proline, Btd, APM, ACTB, or ACDN or have the formulaX-Y-Z wherein X is a single amino acid (all types), preferably Asn, Asp,Ala, His, GlnGly, beta-Alanine), or a short one (Ala, Ser, Thr, Asp),and their N-alkyl, N-hydroxyl, or N-aryl analogs or a synthetic aminoacids, Y is a single amino acids (all types), preferably Pro, Ile, Ala,Val (both D and L) and their N-alkyl, N-hydroxyl, or N-aryl analogs,Synthetic amino acid. Amino acid mimetic which constrain the dihedralangles or the side chain to favorable configurations, Z is a singleamino acid (all types), preferably with no sidechain or a short sidechain and their N-alkyl, N-hydroxyl, or N-aryl analogs, R8 is a Cysteineside chain analog [—(CR2)n-S, or —(CR2)n-H; where n is C1-C10, R is anyatom and H is a reactive group], an alpha, beta or gamma amino acidscapable of forming a crosslink with Cys14 via a side chain functionalgroup, an amino acid mimetic which constrain the dihedral angles or theside chain to favorable configurations and R9 is preferably smaller than1000 daltons.
 14. A method of claim 12 wherein R1 is Y, NTFY, SSSNY,SSDWDY, lb, PN, or QE; R2 is a 2-carboxy-3-bromo-pyridine,2-bromo-benzoic acid, 2-bromo-cyclohexanoic acid, a blocked C, crosslinked to R6 by a Carba link, any non-crosslinking amino acid ormimetic; R3 is 2-carboxy-3-thiopiperidine, or C crosslinked to C at R8;R4 is a sarcosine, ornithine and lysine or having the formula J-Kwherein J is Gln, Gly, Ser, Ala, Pro, Asp, Asn,L-octahydroindole-2-carboxyilic acid, and their N-alkyl, N-hydroxyl, orN-aryl analogs and K is Ile, Norleucine, Met, Val, Phe, Trp, Tyr, Gln,Gly, Ala, Pro and L-1,2,3,4-tetrahydroisoquinoline-3-carboxyilic acid,and their N-alkyl, N-hydroxyl, or N-aryl analogs; R5 is Ala, N(Me)-Ala,Thr, Leu, N(Me)-Leu, Phe blocked Cysteine, Serine or Threonine, orL-1,2,3,4-tetrahydroisoquinoline-3-carboxyilic acid; R6 is C crosslinked to R2, blocked C such as Cmob, cross linked to R2 by a Carba linkany non-crosslinking amino acid or mimetic. R7 is a X-Y-Z wherein X is aIsoasparagine or beta alanine. Y is Pro, Ile, Ala, Val (both D and L)and their N-alkyl, N-hydroxyl, or N-aryl analogs, homopro, Nipecoticacid, isonipecotic acid, Oic, Tic, Aib, aminobenzoate,carboxypiperidine, azetidine carboxylate and aminocyclopentenecarboxylic acid or L-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid; Zis a Gly, beta-Alanine, Ala, Ser, Thr, Asp, and their N-alkyl,N-hydroxyl, or N-aryl analogs; R8 is a 2-carboxy-3-thiopiperidine, Ccrosslinked to C at R3 or any other moiety crosslinked to R3; and R9 is0-20 amino acids.
 15. A compound having the formula of Formula (II):R201-R202-R203-R204-R205-R206-R207 wherein:  (II) R201 is absent orselected from the group consisting of: 1-50 natural amino acids, 1-50blocked amino acids, 1-50 synthetic amino acids, 1-50 derivatized aminoacids; 1-50 amino acid mimetics, a 1-50 alkyl, one or more 1-50substituted alkyls, one or more 1-50 aryls, one or more 1-50 substitutedaryls, one or more 1-50 alkylaryls, one or more 1-50 substitutedalkylaryls and combinations thereof; R202 is absent or selected from thegroup consisting of: a natural amino acid, a blocked amino acid, asynthetic amino acid, a derivatized amino acid, an amino acid mimetic;R203 is absent or selected from the group consisting of: a natural aminoacid, a blocked amino acid, a synthetic amino acid, a derivatized aminoacid, an amino acid mimetic; wherein if R202 crosslinks with R205 orR206, then R203 is absent or does not crosslink to any other amino acidor an amino acid mimetic, and if R202 is absent or does not crosslinkwith R205 or R206, then R203 crosslinks with R205 or R206; R204 isselected from the group consisting of: one or two natural amino acids,one or two blocked amino acids, one or two synthetic amino acids, one ortwo derivatized amino acids, one or two amino acid mimetics, or adipeptide mimetic and combinations thereof, wherein R204 forms a3₁₀-helix turn group linking R203 to R205; R205 is absent or selectedfrom the group consisting of: a natural amino acid, a blocked aminoacid, a synthetic amino acid, and a derivatized amino acid and an aminoacid mimetic, R206 is absent or selected from the group consisting of: anatural amino acid, a blocked amino acid, a synthetic amino acid, aderivatized amino acid and an amino acid mimetic acid, wherein if R205crosslinks with R202 or R203, then R206 is absent or does not crosslinkto any other amino acid or an amino acid mimetic, and if R205 is absentor does not crosslink with R202 or R203, then R206 crosslinks with R202or R203; and R207 is absent or selected from the group consisting of:1-50 natural amino acids, 1-50 blocked amino acids, 1-50 synthetic aminoacids, 1-50 derivatized amino acids; 1-50 amino acid mimetics, a 1-50alkyls, one or more 1-50 substituted alkyls, one or more 1-50 aryls, oneor more 1-50 substituted aryls, one or more 1-50 alkylaryls, one or more1-50 substituted alkylaryls, and combinations thereof.
 16. The compoundof claim 15 wherein R201 is absent or tyrosine.
 17. The compound ofclaim 15 wherein R202 is cysteine or penacillamine.
 18. The compound ofclaim 15 wherein R203 is serine, alanine or blocked cysteine.
 19. Thecompound of claim 15 wherein R204 is E-L.
 20. The compound of claim 15wherein R205 is alanine or blocked cysteine.
 21. The compound of claim15 wherein R206 is cysteine or penacillamine.
 22. The compound of claim15 wherein R207 is absent, or NPAAAGCacmY.
 23. The compound of claim 15wherein said compound is YCSELAC or CAELCacmCNPAAAGCacmY.
 24. A compoundhaving Formula (III):R301-R302-R303-R304-R305-R306-R307-R308-R309   wherein: R301 is absentor selected from the group consisting of: 1-50 natural amino acids, 1-50blocked amino acids, 1-50 synthetic amino acids, 1-50 derivatized aminoacids; 1-50 amino acid mimetics, a 1-50 alkyls, one or more 1-50substituted alkyls, one or more 1-50 aryls, one or more 1-50 substitutedaryls, one or more 1-50 alkylaryls, one or more 1-50 substitutedalkylaryls, and combinations thereof; R302 is absent or selected fromthe group consisting of: a natural amino acid, a blocked amino acid, asynthetic amino acid, a derivatized amino acid, and an amino acidmimetic; wherein R302 may crosslink with R306; R303 is selected from thegroup consisting of: a natural amino acid, a blocked amino acid, asynthetic amino acid, a derivatized amino acid and amino acid mimetic;wherein R303 crosslinks with R308; R304 is selected from the groupconsisting of: one or two natural amino acids, one or two blocked aminoacids, one or two synthetic amino acids, one or two derivatized aminoacids, one or two amino acid mimetics, a dipeptide mimetic, andcombinations thereof; wherein R304 forms a 3₁₀-helix turn group linkingR303 to R305; R305 is selected from the group consisting of: a naturalamino acid, a blocked amino acid, a synthetic amino acid, a derivatizedamino acid, and an amino acid mimetic; wherein R305 does not crosslinkwith R309; R306 is selected from the group consisting of: a naturalamino acid, a blocked amino acid, a synthetic amino acid, a derivatizedamino acid, and an amino acid mimetic; wherein R306 may crosslink withR302; R307 is selected from the group consisting of: one, two or threenatural amino acids, one, two or three blocked amino acids, one, two orthree amino acid mimetics, or a tripeptide mimetic and combinationsthereof; wherein R307 forms a beta turn group linking R306 to R308; R308is selected from the group consisting of: a natural amino acid, ablocked amino acid, a synthetic amino acid, a derivatized amino acid,and an amino acid mimetic; wherein R308 crosslinks with R303; and R309is absent or selected from the group consisting of: 1-50 natural aminoacids, 1-50 blocked amino acids, 1-50 synthetic amino acids, 1-50derivatized amino acids; 1-50 amino acid mimetics, a 1-50 alkyls, one ormore 1-50 substituted alkyls, one or more 1-50 aryls, one or more 1-50substituted aryls, one or more 1-50 alkylaryls, one or more 1-50substituted alkylaryls and combinations thereof; wherein R309 does notcrosslink to R305; and wherein the structure of the compounds is not:C-hC-E-L-A-C-N-P-A-X; hCC-E-L-A-X-N-P-A-C;C-C-E-L-A-C-N-P-A-C;C-C-E-L-A-C-N-P-A-C-T-G-A;Y-Ca-C-E-L-F-Ca-N-P-A-C;Y-C-C-E-L-mA-C-N-P-A-C;Y-C-C-E-mL-A-C-N-P-A-C;C-C-E-L-Cm-C-N-P-A-C-A-G-Cm;C-C-E-L-Ca-C-N-P-A-C-A-G-Ca; orX-Ca-E-L-A-hC-N-P-A-Ca; wherein hC is Homocysteine; X is Alanine linkedto hC via a covalent CH2-S bond between an Ala side chain and an hC sidechain; Cm is Cysteine(S-methoxybenzyl); and Ca isCysteine(S-acetamidomethyl).
 25. A compound having Formula (IV):R401-R402-R403-R404-R405-R406-R407-R408-R409  (IV) wherein: R401 isabsent of selected from the group consisting of: 1-50 natural aminoacids, 1-50 blocked amino acids, 1-50 synthetic amino acids, 1-50derivatized amino acids; 1-50 amino acid mimetics, a 1-50 alkyls, one ormore 1-50 substituted alkyls, one or more 1-50 aryls, one or more 1-50substituted aryls, one or more 1-50 alkylaryls, one or more 1-50substituted alkylaryls and combinations thereof; R402 is absent orselected from the group consisting of: a natural amino acid, blockedamino acid, synthetic amino acid, derivatized amino acid, and an aminoacid mimetic; wherein R402 may crosslink with R406; R403 is absent orselected from the group consisting of: a natural amino acid, blockedamino acid, synthetic amino acid, derivatized amino acid and amino acidmimetic; wherein R403 crosslinks with R408; R404 is selected from thegroup consisting of: one or two natural amino acids, one or two blockedamino acids, one or two synthetic amino acids, one or two derivatizedamino acids, one or two amino acid mimetics, or a dipeptide mimetic andcombinations thereof; wherein R404 forms a 3₁₀-helix turn group linkingR403 to R405; R405 is selected from the group consisting of: a naturalamino acid, blocked amino acid, synthetic amino acid, derivatized aminoacid and amino acid mimetic; wherein R405 does not crosslink with R409;R406 is selected from the group consisting of: a natural amino acid,blocked amino acid, synthetic amino acid, derivatized amino acid, andamino acid mimetic; wherein R406 may crosslink with R402; R407 isselected from the group consisting of: one, two or three natural aminoacids, one, two or three blocked amino acids, one, two or three aminoacid mimetics, or a tripeptide mimetic and combinations thereof; whereinR407 forms a beta turn group linking R406 to R408 R408 is selected fromthe group consisting of: a natural amino acid, blocked amino acid,synthetic amino acid, derivatized amino acid and amino acid mimetic;wherein R408 crosslinks with R403; R409 is absent; wherein the structureof the compounds is not:C-hC-E-L-A-C-N-P-A-X-hCC-E-L-A-X-N-P-A-C;C-C-E-L-A-C-N-P-A-C;Y-Ca-C-E-L-F-Ca-N-P-A-C;Y-C-C-E-L-mA-C-N-P-A-C;Y-C-C-E-mL-A-C-N-P-A-C;C-C-E-L-Cm-C-N-P-A-C-A-G-Cm;C-C-E-L-Ca-C-N-P-A-C-A-G-Ca; orX-Ca-E-L-A-hC-N-P-A-Ca; wherein hC is Homocysteine; X is Alanine linkedto hC via a covalent CH2-S bond between an Ala side chain and an hC sidechain; Cm is Cysteine(S-methoxybenzyl); and Ca isCysteine(S-acetamidomethyl).
 26. A compound having Formula (V):R501-R502-R503-R504-R505-R506-R507-R508-R509  (V) wherein: R501 isabsent of selected from the group consisting of: 1-50 natural aminoacids, 1-50 blocked amino acids, 1-50 synthetic amino acids, 1-50derivatized amino acids; 1-50 amino acid mimetics, a 1-50 alkyls, one ormore 1-50 substituted alkyls, one or more 1-50 aryls, one or more 1-50substituted aryls, one or more 1-50 alkylaryls, one or more 1-50substituted alkylaryls and combinations thereof; R502 is absent orselected from the group consisting of: a natural amino acid, blockedamino acid, synthetic amino acid, derivatized amino acid, and an aminoacid mimetic; wherein R502 may crosslink with R506; R403 is absent orselected from the group consisting of: a natural amino acid, blockedamino acid, synthetic amino acid, derivatized amino acid and amino acidmimetic; wherein R503 crosslinks with R508; R504 is selected from thegroup consisting of: one or two natural amino acids, one or two blockedamino acids, one or two synthetic amino acids, one or two derivatizedamino acids, one or two amino acid mimetics, or a dipeptide mimetic andcombinations thereof; wherein R504 forms a 3₁₀-helix turn group linkingR503 to R505; R505 is selected from the group consisting of: a naturalamino acid, blocked amino acid, synthetic amino acid, derivatized aminoacid and amino acid mimetic; wherein R505 does not crosslink with R509;R506 is selected from the group consisting of: a natural amino acid,blocked amino acid, synthetic amino acid, derivatized amino acid, andamino acid mimetic; wherein R506 may crosslink with R502; R507 isselected from the group consisting of: one, two or three natural aminoacids, one, two or three blocked amino acids, one, two or three aminoacid mimetics, or a tripeptide mimetic and combinations thereof; whereinR507 forms a beta turn group linking R506 to R508 R508 is selected fromthe group consisting of: a natural amino acid, blocked amino acid,synthetic amino acid, derivatized amino acid and amino acid mimetic;wherein R508 crosslinks with R503; R509 is selected from the groupconsisting of: 1-50 natural amino acids, 1-50 blocked amino acids, 1-50synthetic amino acids, 1-50 derivatized amino acids; 1-50 amino acidmimetics, a 1-50 alkyls, one or more 1-50 substituted alkyls, one ormore 1-50 aryls, one or more 1-50 substituted aryls, one or more 1-50alkylaryls, one or more 1-50 substituted alkylaryls and combinationsthereof; wherein R509 does not crosslink to R505; wherein the compoundis degradable in vivo and the structure of the compounds is notC-C-E-L-A-C-N-P-A-C-T-G-A.
 27. A compound having Formula (VI):R601-R602-R603-R604-R605-R606-R607-R608-R609  (VI) wherein: R601 isabsent of selected from the group consisting of: 1-50 natural aminoacids, 1-50 blocked amino acids, 1-50 synthetic amino acids, 1-50derivatized amino acids; 1-50 amino acid mimetics, a 1-50 alkyls, one ormore 1-50 substituted alkyls, one or more 1-50 aryls, one or more 1-50substituted aryls, one or more 1-50 alkylaryls, one or more 1-50substituted alkylaryls and combinations thereof; R602 is absent orselected from the group consisting of: a natural amino acid, blockedamino acid, synthetic amino acid, derivatized amino acid, and an aminoacid mimetic; wherein R602 may crosslink with R606; R603 is absent orselected from the group consisting of: a natural amino acid, blockedamino acid, synthetic amino acid, derivatized amino acid and amino acidmimetic; wherein R603 crosslinks with R608; R604 is selected from thegroup consisting of: one or two natural amino acids, one or two blockedamino acids, one or two synthetic amino acids, one or two derivatizedamino acids, one or two amino acid mimetics, or a dipeptide mimetic andcombinations thereof; wherein R604 forms a 3₁₀-helix turn group linkingR603 to R605; R605 is selected from the group consisting of: a naturalamino acid, blocked amino acid, synthetic amino acid, derivatized aminoacid and amino acid mimetic; wherein R605 does not crosslink with R609;R606 is selected from the group consisting of: a natural amino acid,blocked amino acid, synthetic amino acid, derivatized amino acid, andamino acid mimetic; wherein R606 may crosslink with R602; R607 isselected from the group consisting of: one, two or three natural aminoacids, one, two or three blocked amino acids, one, two or three aminoacid mimetics, or a tripeptide mimetic and combinations thereof; whereinR607 forms a beta turn group linking R606 to R608 R608 is selected fromthe group consisting of: a natural amino acid, blocked amino acid,synthetic amino acid, derivatized amino acid and amino acid mimetic;wherein R608 crosslinks with R603; R609 is selected from the groupconsisting of: 1-50 natural amino acids, 1-50 blocked amino acids, 1-50synthetic amino acids, 1-50 derivatized amino acids; 1-50 amino acidmimetics, a 1-50 alkyls, one or more 1-50 substituted alkyls, one ormore 1-50 aryls, one or more 1-50 substituted aryls, one or more 1-50alkylaryls, one or more 1-50 substituted alkylaryls and combinationsthereof; wherein R609 does not crosslink to R605 and is non-degradablein vivo.
 28. A conjugated compound that comprises a moiety of a formulaselected from the group consisting of: Formula (I), Formula (II),Formula (III), Formula (IV), Formula (V), and Formula (VI), that isconjugated to an active moiety which may be a radionuclide, an enzyme, afluorescent label, a metal chelating group, a chemiluminescent label, abioluminescent label, a chemotherapeutic, a toxin, an inactive prodrug,a radiosensitizing agent, a photodynamic agent, a nucleic acid moleculeor combinations thereof.
 29. A composition comprising liposomes thatcomprise a compound of claim 15-28 in combination with an active agentselected from the group consisting of: a radionuclide, an enzyme, afluorescent label, a metal chelating group, a chemiluminescent label, abioluminescent label, a chemotherapeutic, a toxin, an inactive prodrug,a radiosensitizing agent, a photodynamic agent, a nucleic acid moleculeor combinations thereof.
 30. A composition comprising a compound ofclaim 15-28 in combination with a chemotherapeutic, a toxin, orcombinations thereof.
 31. A method of diagnosing cancer characterized byexpression of GCC in an individual comprising the step of detecting GCCin an extraintestinal sample from an individual by contacting the sampleor portions thereof with compound of claim 15-28 and detecting thepresence of the compound bound to the sample.
 32. A method of imagingcancer characterized by expression of GCC in an individual comprisingthe step of administering to the individual a conjugated compound thatcomprises a compounds of claim 28 in which active moiety of theconjugated compound is a detectable label, wherein accumulation of theconjugated compound at a site on the individual's body is detected. 33.A method of treating cancer characterized by expression of GCC in anindividual comprising the step of administering to the individual such acompound of claim 15-28 kills or inhibits replication of cells to whichit binds.
 34. The present invention further relates to method oftreating an individual who has enterotoxin mediated diarrhea or who isat risk of contracting enterotoxin mediated diarrhea comprising the stepof administering to said individual a compound with a structureaccording to Formula (II) or Formula (VI).
 35. A method of treating anindividual who has enterotoxin mediated diarrhea or who is at risk ofcontracting enterotoxin mediated diarrhea. The methods comprise the stepof administering to said individual a compound with a structureaccording to Formula (1), Formula (III), Formula (IV) or Formula (V)under conditions such that the C terminal is not degradable such thatthe compound is converted to an agonist.
 36. A method of treating anindividual who has cancer or who is at risk of developing cancercomprising the step of administering to said individual a compound witha structure according to Formula (I) Formula (III), Formula (IV) orFormula (V) under conditions such that the C terminal is degradable suchthat the compound is converted to an agonist.
 37. The method of claim 36wherein the compound is administered in combination with achemotherapeutic agent or a toxin.
 38. A method of treating anindividual who has cancer or who is at risk of developing cancercomprising the step of administering to said individual a compound witha structure according to Formula (II).
 39. The method of claim 38wherein the compound is administered in combination with achemotherapeutic agent or a toxin.